Synthetic antibody mimetic compounds (syams) targeting cancer, especially prostate cancer

ABSTRACT

The present invention relates to compounds which function as antibody mimetic compounds. These compounds are bifunctional/multifunctional compounds which contain at least one cancer cell binding moiety which selectively binds to prostate specific membrane antigen (PSMA) and a FC receptor binding moiety which modulates an FC immune receptor, preferably a FcγRI receptor. Compounds according to the present invention bind selectively to cancer cells which upregulate PSMA and through that interaction, place the Fc receptor binding moiety of the compound in proximity to a Fc receptor, preferably a FcγRI receptor, which can modulate (preferably, upregulate) a humoral response in a patient to cancer cells. Through this biological action of the compounds according to the present invention, cancer cells, including metastatic cancer cells, especially prostate cancer cells can be immune regulated, resulting in the favorable therapy of cancer in a patient. Methods of using these compounds to treat cancer and/or reduce the likelihood of metastatis of cancer are additional aspects of the present invention.

GRANT SUPPORT

This invention was supported by grant no. IDP2OD002913-01 from theNational Institutes of Health. Consequently, the government retainscertain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to compounds which function as syntheticantibody mimetic compounds. Compounds according to the present inventionare bifunctional/multifunctional compounds which contain at least onecancer cell binding moiety which selectively binds to prostate specificmembrane antigen (PSMA) and a FC receptor binding moiety which modulatesan FC immune receptor, preferably a FcγRI receptor. Compounds accordingto the present invention bind selectively to cancer cells whichupregulate PSMA and through that interaction, place the Fc receptorbinding moiety of the compound in proximity to a Fc receptor, preferablya FcγRI receptor, which can modulate (preferably, upregulate) a humoralresponse in a patient to cancer cells. Through this biological action ofthe compounds according to the present invention, cancer cells,including metastatic cancer cells, especially prostate cancer cells canbe immune regulated, resulting in the favorable therapy of cancer in apatient. Methods of using these compounds to treat cancer and/or reducethe likelihood of metastatis of cancer are additional aspects of thepresent invention.

BACKGROUND OF THE INVENTION

A growing wealth of data indicates that targeted therapies can mobilizea patient's own immune system to destroy malignancies with fewer sideeffects than traditional chemotherapy. Since it is estimated that 41% ofAmericans—almost 1 in 2 people—born in 2011 will develop cancer in theirlifetime, the generation of more effective cancer immunotherapies is ahigh priority. These therapies include monoclonal antibodies (mAbs) thatdirect innate immune cells to tumor-associated antigens (TAA) as well ascancer “vaccines” that take many forms (including injections of tumorproteins with adjuvants or ex vivo primed dendritic cells) and aredesigned with the intention of inducing long-lasting anti-tumor T-cells.

The objective of the present inventors' research is to develop novelcompounds capable of stimulating immune responses against tumors. Theintroduction of monoclonal antibodies (mAbs) has revolutionized thefield of immunotherapy, particularly cancer therapy. Although mAbs havebecome a mainstay of cancer therapeutics, they possess seriousdrawbacks.¹ mAbs are limited by their dangerous immunologicalside-reactions, lack of oral bioavailability, and high cost ofproduction and administration. Development of synthetic molecules thatmimic antibody function may provide an effective solution for theaforementioned problems.

Current research is exploring the development of new therapeutics thattake advantage of the immune system's natural responses. Optimization ofthe Fc region of unconjugated monoclonal antibodies to increase theirefficacy and response, has been of great interest.² ³ One recentlydeveloped derivative of monoclonal antibodies, bispecific antibodies,ligates two Fabs with different target specificity. One Fab region bindsto the target protein of interest and the other binds to an immunereceptor of choice^(4,5,) including FcγRI ⁶, e.g., bispecific antibodiestargeting HER2 and FcγRI. ⁶ In an alternative approach, the presentinventors, and others, have utilized rational design to constructsynthetic systems capable of performing, or templating, compleximmunological functions.⁷

As a result, several antibody-recruiting molecules (ARMs) that canmodulate the immune system⁸ have occurred. ARMs are bifunctionalsynthetic molecules that contain a target-binding terminus (TBT), whichbinds to pathogenic surface proteins with high affinity and specificity,and an antibody-binding terminus (ABT) that recruits endogenousantibodies. We have shown that these molecules are capable of elicitinga targeted immune response selectively against both cancer and virusinfected cells. This topic has been reviewed recently.⁴

Notwithstanding the development of ARMs, the manipulation of the immunesystem with fully synthetic molecules is currently in its infancy.⁹ Inthe present invention, a relatively small molecular weight antibodymimic can perform both targeting and immune effector functions, anapproach which holds greater promise for the treatment of cancer,especially prostate cancer. For the development of this fully syntheticantibody mimetic, we chose prostate cancer as our pathogenic target,although the approach can be used anywhere PSMA is expressed, includingvirtually all cancers, but especially prostate cancer and metastaticprostate cancer.

The choice of prostate cancer as a target for the development of thepresent invention reflects its severity in causing disease and death.Prostate cancer is the second leading cause of cancer related deathsamong American males, and current strategies for treatment often leadsto relapse and undesirable side effects.¹⁰ It has been predicted thatone out of every six American men will develop prostate cancer duringtheir lifetime. Currently, there are no clinically approved monoclonalantibody-based drugs targeting prostate cancer. Obviously, animmunological approach to the treatment of prostate cancer represents anapproach with great potential, however, the negative attributes of thepresent immunological approaches must be ameliorated for this generalapproach to be successful. The present invention represents analternative approach to address these problems.

THE PRESENT INVENTION

The current work which is presented in this application has led to theinventive development of a fully synthetic functional mimetic of anantibody that can overcome some of the drawbacks that are limiting thetherapeutic potential of monoclonal antibodies. This new approachleverages the advantages of traditional small molecules with those ofnext-generation biologics to address the current limitations. Here, wereport that a rationally designed synthetic molecule, called “syntheticantibody mimetic targeting prostate cancer” (SyAM-P), which is capableof redirecting the immunological functions of FcγRI towards targetsdisplaying prostate specific membrane antigen (PSMA), thus targeting anderadicating cancer cells which upregulate PSMA, including prostatecancer cells, including metastatic prostate cancer cells. The inventorshere present the first examples of synthetic molecules that display theproperties and functions of an antibody, which entails the selectivetargeting of an immune response against a pathogenic target, in thiscase cancer cells, including metastatic cancer cells, especiallyprostate cancer cells and metastatic prostate cancer cells.

The present invention relates to compounds which are designated SyAM-Ps.SyAM-Ps are multifunctional small molecules designed to stimulate bothinnate and adaptive anti-tumor immune responses, especially immuneresponses which are modulated through FcγRI. The inventors havepreviously developed bifunctional antibody recruiting molecules (ARMs)able to redirect endogenous antibodies to prostate cancer cellsexpressing prostate specific membrane antigen (PSMA). The presentinvention enhances the immunostimulatory properties of ARMs by attachingmodulators of FcγRI (potent modulators of humoral response) to the ARMscaffold. The binding moieties from the parent compound target thecancer cells which exhibit an upregulation of PSMA, in particular,prostate cancer cells, while the additional FcγRI motif activates alocal humor response for induction of immunologic memory against thetumor. The result is an effect which provides synergistic anticanceractivity which is substantially greater than the anticancer activity ofindividual functional molecules which are not linked together as in thepresent invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows A) depiction of proposed mechanism of action of thesynthetic antibody mimetics; B) shows a modeling of membrane distancesand length requirements during the interaction between a monoclonalantibody against PSMA binding to an Fc receptor; and C) shows aschematic of evolution of design of SyAM-P from monoclonal antibodytemplate.

FIG. 2 exemplifies compounds and related moieties according to thepresent invention. CP33 is a FcγRI targeting motif 1, SyAM-P1, firstgeneration displaying a single FcγRI and PSMA binding motifs linked withaminocaproic units. 2, SyAm-P2, second generation displaying a pair ofPSMA targeting motifs linked to a single CP33 motif. 3, SyAM-P3, thirdgeneration displaying a pair of PSMA targeting motifs linked to a singleCP33 motif. The biotin moiety is present as a handle so that thecompounds may be readily isolated or identified in experiments which areotherwise disclosed herein. In practice, therapeutic compounds accordingto the present invention avoid the inclusion of a biotin molecule andthe compounds contain a hydrogen, alkyl group or an acyl group at theamine group bonded to the biotin moiety depicted in the figure. 3,SyAm-P3, third generation displaying a pair of PSMA targeting motifslinked to a pair of CP33 targeting moieties/molecules.

FIG. 3 shows A) binding of compound 1 to PSMA expressing RM1.PGLS cellsin a dose dependent manner; B) binding of 1 (SyAm-P1) to IIA1.6 cellsexpressing FcγRI in a dose dependent manner; C) ability of 1 to templatea ternary complex between IIA1.6 cells expressing FcγRI and solublerecombinant human PSMA; D) superoxide burst generation against PSMAlabeled beads by IFN-γ primed U937 cells induced by 1. Phagocytosis andsuperoxide burst data is representative experiments performed intriplicate on three separate occasions; E) phagocytosis using IFN-γprimed U937 cells as effectors and fluorescent polystyrene PSMA labeledbeads induced by 1 in a dose dependent and PSMA dependent manner.

FIG. 4 shows A) comparison of phagocytosis induced by compound 2(SyAMP-2) and 3 (SyAMP-3) against PSMA labeled beads by IFN-γ primedU937 cells. A dose and PSMA dependent response is seen with an increasein efficacious range and level by compound 3 as compared to compound 2;B) comparison of superoxide burst by primed U937 cells against PSMAlabeled beads in a dose dependent manner by compounds 2 and 3 with anincrease in range and amount of compound 3; C) phagocytosis of PSMAexpressing RM1.PGLS cells as induced by compound 3 using IFN-γ; D) amnisflow cytometry imaging of phagocytosis with representative images ofcompleted phagocytosis with representative images of completedphagocytosis as compared to phagocytic cup formation. Channels shown arebrightfield, target (stained with F11 dye DiO), nuclide (stained DAP1),macrophage (stage F12 DID) and merged image.

FIG. 5, Table 1, shows phagocytosis of PSMA coated 6 μm beads by IFN-γprimed U937 cells in the presence of 50 nM of compound 2. The tablelists the measured PSMA per square μm using phycoerythrin labeledanti-PSMA antibody for 8.2 pmol/bead. Calculated PSMA per square μm for5.7 pmol/bead and 2.9 pmol/bead on loading capacity compared to 8.2pmol/bead. PSMA measurement of RM1.PGLS cells using phycoerythrinanti-PSMA antibody. Phagocytosis of targets performed with primed U937cells and 50 nM compound 2.

FIG. 6 shows phagocytosis of PSMA labeled 6 μm beads by IFN-γ primedU937 cells. Comparison of phagocytic response induced by variousconcentrations of either compound 1 or compound 2.

FIG. 7 shows A) structures of different ligands connecting the urea PSMAbinding moieties differing in length and hydrophobicity; B) showsphagocytosis of PSMA labeled 6 μm beads by IFN-γ primed U937 cells.Comparison of phagocytic response induced by various concentrations ofSyAMs possessing a pair of PSMA binding motifs linked to CP33 withvarious linker lengths and compositions.

FIG. 8 shows A) prozone phenomenon where excess concentration causes areduction in Fc receptor SyAM-P binding (even as SyAM-P with PSMAremains intact); B) shows an overlay of data fit to analytical ternarycomplex model. Increase in efficacy and potency consistent with a factorof 5 increase to target affinity from SyAM-P1 to SyAM-P2. Observedincrease of efficacy and potency consistent with two order of magnitudeincrease from SyAM-2 to SyAM-P3. Increase for SyAM-P3 due to improvementof weaker binding affinity having a larger net effect in the analyticalmodel.

FIG. 9 shows phagocytosis of PSMA coated with 6 μm beads by IFN-γ primedby U937 cells in the presence of 50 nM of SyAM-P3. A) shows inhibitionof phagocytosis by increasing concentrations (in NM) by human IgG,inhibiting the interaction of the molecule with the Fc receptor. B)shows inhibition of phagocytosis by increasing concentrations of 2-PMPA,inhibiting the interaction between the molecule and PSMA.

FIG. 10 shows A) the area under the curve (AUC) of superoxide burstassay comparing +/− target PSMA+ beads in the presence of variousconcentrations of SyAM-P3. Little to no accumulation of superoxide seenover assay time. B) shows peak superoxide production time taken (xmin)again little to no background superoxide detection in the presence ofSyAM-P3 alone.

FIG. 11 shows phagocytosis of PSMA expressing RM1.PGLS cells by IFN-γprimed U937 cells in the presence of 6.25 nM of SyAM-P3. A) shows theinhibition of phagocytosis by increasing concentrations (in nM) by humanIgG, inhibiting the interaction of the molecule with Fc receptor. B)shows the inhibition of phagocytosis by increasing concentrations of2-PMPA, inhibiting the interaction between the molecule and PSMA.

FIG. 12 shows phagocytosis of RM1.PGLS cells by Fn-γ primed U937 cellsin the presence of various concentrations of either compound 3 or ArmP8with anti-DNP antibody (133 nM). Phagocytosis subtracted from backgroundof no molecule. Bars for compound 3 evidence phagocytosis atsignificantly less concentration than for ArmP8.

FIG. 13, Table 2 shows Amnis phagocytosis: attached double positivesrefer to all events scored as ADCP in traditional FACS where targets andeffectors are stained two different colors. All in-focus eventscollected by Amnis imagestreamflow cytometry were then classified as“completed” (where a target is entirely engulfed by a macrophage) or as“initiated” (where a clear phagocytic cup had formed between macrophageand target).

FIG. 14 shows a number of Fc receptor binding moieties which exhibitactivity as modulators of FcγRI and can be used as an [IBT] grouppursuant to the present invention. In the figure, each of the compoundsis shown with an attachment bond at a free hydroxyl (OH group) or amine(NH2).

FIG. 15 provides a scheme for the chemical synthesis of SyAm-P1. Thissynthesis is discussed in the text of the specification in the chemicalsynthesis section.

FIG. 16 shows the final product SyAM-P1, synthesized by the methodpresented in FIG. 15. It is noted that in diagnostic applications, thelysine amino acid and biotin covalently bonded to the CP33 moiety may bereadily replaced by any number of amino acids which have unreactiveamino acid side chains.

FIG. 17 shows the chemical synthesis of SyAM-P2 which contains a biotingroup for diagnostic applications.

FIG. 18 shows the chemical synthesis (urea formation) and linkersynthesis for certain parts of SyAM-P3.

FIG. 19 shows the chemical synthesis of the second linker synthesis forSyAM-P3 which contains a [MULTICON] group.

FIG. 20 shows the chemical synthesis to link the CBT groups to the[MULTICON] group containing an azide for further modification to provideSyAM-P3. The azide is subsequently shown being reacted with the complexacetylenic compound containing two free amine groups which are linkedthrough to the triazole group formed when the acetylenic moiety reactswith the azide moiety to form the complex intermediate which can becondensed with two CP33 groups to form SyAM-P3.

FIG. 21 shows the introduction of a diketo linker group onto theCP33-lysine intermediate (the carboxylic acid group of the lysine havingbeen end-capped with an amine to form an amide group), the linker beingend-capped with succinimide leaving groups. The final intermediate,pictures, contains a succinimide leaving group at the distill end of thediketo linker which can be reacted with the two free amine groups of theintermediate from FIG. 20 to provide the final product SyAM-P3 of FIG.22.

FIG. 22 shows the final product SyAM-P3.

FIG. 23 shows an alternative linker strategy for the synthesis ofSyAm-P2 compounds. The scheme shows the synthesis of SyAM-P2 usingaminohexanoic acid polypeptide linkers, polyethylene glycol linkers andpolyethylene glycol linkers which are further linked into extendedlinkers through an amide group.

FIG. 24 shows chemical synthesis steps for SyAM-P2 using alternativelinkers.

OBJECTS OF THE INVENTION

It is an object of the invention to provide chimeric bifunctional andmultifunctional compounds which can be used to treat virtually anycancer (including metastatic cancer), especially including prostatecancer and metastatic prostate cancer.

It is another object of the invention to provide compounds which containa cell binding moiety which binds to prostate specific membrane antigen(PSMA) of cancer cells, especially prostate cancer cells and metastaticprostate cancer cells and an Fc receptor binding moiety which binds toFc receptors, in particular FcγRI receptors, and modulates ahumoral/antibody response to cancer cells to which the compounds bind inorder to cause cancer cell death and to treat cancer.

It is an additional object of the invention to provide chimericbifunctional and multifunctional compounds which can be used to providepharmaceutical compositions, including pharmaceutical compositions whichinclude additional bioactive agents or agents which assist in thetreatment of cancer, especially prostate cancer, including metastaticprostate cancer.

It is still another object of the invention to provide methods fortreating cancer, especially prostate cancer, including metastaticprostate cancer using chimeric bifunctional and multifunctionalcompounds according to the present invention which exhibit unexpectedand synergistic anticancer activity.

Yet a further object of the invention is to provide methods forinhibiting and/or reducing the likelihood of metastatis of cancer,especially including metastatic prostate cancer.

These and/or other objects of the invention may be readily gleaned froma review of the invention as described herein.

BRIEF DESCRIPTION OF THE INVENTION

In one embodiment, the invention provides bifunctional ormultifunctional compounds which bind to prostate specific membraneantigen (PMSA) on cancer cells and separately, to a Fc receptor, often aFcγRI receptor through the [IBT] group. Through modulation of the Fcreceptor (often a Fcγ receptor, most often a FcγRI receptor) thecompound, situated on the surface of a cancer cell (often a prostatecancer cell and/or a metastatic cancer cell) stimulates immune effectorcells, thus increasing immune signaling and phagocytic and/or cytotoxicresponses acting in a synergistic manner to assist in eliminating cancercells from tissue, thus treating the disease.

In one embodiment, the present invention relates to compounds accordingto the general formula:

where [IBT] is an Fc (often a Fcγ receptor, most often a FcγRI receptor)receptor binding moiety;[CBT] is a cell binding moiety which binds to prostate specific membraneantigen (PSMA);L₁ and L₂ are linker groups (which groups may include one or morebifunctional connector groups [CON} or more than one linker to providean extended linker group);[MULTICON} is a bifunctional or multifunctional connector group(preferably, multifunctional) which, when present, connects at least one[IBT) group to at least one [CBT] group through a linker;MCON is an integer from 0 to 10, often 1 to 10, more often 1 to 5, often0, 1 or 2;n and n′ are each independently n integer from 1 to 15, often 2 to 10,often 2 to 5, more often 2 or 3, or 2, 3, 4 5 or 6;NL1 and NL2 are each an integer from 0 to 10, often 1 to 10, often 2 to5, more often 2 or 3, with the proviso that n≧NL1 and n′≧NL2.

In another embodiment, [IBT] is CP33 (see FIG. 2 and FIG. 14), whichexhibits excellent activity as a modulator of FCγRI receptors and isparticularly effective at enhancing immune effector cells (e.g.,macrophages and eosinophils) to provide a selective phagocytic and/orcytotoxic response, including opsonization, of the cancer cells,especially prostate cancer, resulting in the eradication of cancer cellsand consequently, cancer tissue. In addition, compounds according to thepresent invention are believed to induce long term immunity againstcancer tissue in the patient, thus reducing the likelihood of a relapseof cancer in a patient who has seen a cancer remission. Thus, compoundsaccording to the present invention may be used in the treatment andprevention of cancer and metastatic cancer, especially includingprostate cancer and metastatic prostate cancer. Compounds according tothe present invention will often contain at least one [IBT] group and atleast two [CBT] groups, often at least two [IBT] groups and two [CBT]groups and often two or three [IBT] groups and two or three [CBT]groups.

Additional compounds according to the present invention includecompounds according to the chemical structure:

Where R₁ is a [IBT] group, often CB33;R₂ is a [CBT] group; often

where k is 4, which is optionally directly linked to a connector group[CON], often a triazole connector group at the ring nitrogen

Where CL is

m is an integer from 0 to 12, often 0, 1, 2, 3, 4, 5, 6and iL is 0 or 1, often 1;X is a [MULTICON] group as otherwise described herein, often a group

where Y₄ is C—H or N; andEach X″ is independently derived from an electrophilic or nucleophilicgroup, preferably (CH₂)_(n″)O, (CH₂)_(n″)N^(RCON), (CH₂)_(n″)S,(CH₂)_(n″) or (CH₂)_(n″)C═O or one or more of X″ is a [CON] group, oftena

group linked through the amine to the ring structure of [MULTICON] whereCL is the same as above;the substitutent RCON is H or a C₁-C₃ alkyl, preferably H or CH₃ andn″ is 0, 1, 2 or 3. andL is a linker group as otherwise described herein, often a group (seeFIG. 2, compound 1, 2, or 3 which links a [CBT] moiety to a [MULTICON]molecule) according to the chemical structure:

where R_(a) is H or CH₃, most often H;m is an integer from 1 to 12, often 1, 2, 3, 4, 5, or 6;m″ is an integer 1, 2, 3, 4, 5, or 6, often 6;t is 0, 1, 2, 3, 4, 5, or 6; andiL is 0 or 1, often 1, wherein L may be linked to a [CON] group and a[CBT] group at one end and a [MULTICON] group on the other end; orAlternatively, L above, links an [IBT] group to a [MULTICON] molecule(as in compound 1 of FIG. 2) either directly or through at least oneamino acid (often, an oligopeptide containing from 1 to 10 amino acidgroups, often lysine or a glycine lysine dipeptide as depicted incompound 1 of FIG. 2), orAlternatively, in the case of a Linker L linking [IBT] to a [MULTICON]group (as in compound 2 of FIG. 2), the linker L is a peptide linkercomprising from 1 to 10 peptides, often a lysine amino acid or adilysine oligopeptide (the free carboxylic acid and amine of lysine maybe end-capped with an amine in the case of the carboxylic acid or anacyl group in the case of a free amine) or other group to preventfurther reactivity); orAlternatively, in the case of a Linker L linking more than one [IBT]group to a [MULTICON] Group, often L is a complex linker group (asdepicted in compound 3 of FIG. 2) which is made up of an ethylene oxidecontaining amine group

where q is 1, 2, 3 or 4 and q′ is 1 to 12, often 1, 2, 3, 4, 5 or 6,the keto group is linked to an amino acid groupwhich is linked to [IBT], often CP33, through a diketo group

and an amino acid often lysine, or an amino acid dipeptide, oftendilysine, one of which lysines is directly bonded to CP33, and anotheramino acid (often lysine) which links the above ethylene oxide aminegroup to a [MULTICON] group through a [CON] group, most often atriazole.

In other embodiments, compounds according to the present invention maybe represented by the structures:

Where R₁ is a [IBT] group, often CB33;R₂ is a [CBT] group; often

where k is 4, which is optionally directly linked to a connector group[CON], often a triazole connector group at the ring nitrogenX is a [MULTICON] group as otherwise described herein, often a group

where Y₄ is C—H or N; andEach X″ is independently derived from an electrophilic or nucleophilicgroup, preferably(CH₂)_(n″)O, (CH₂)_(n″)N^(RCON), (CH₂)_(n″)S,(CH₂)_(n″) or (CH₂)_(n″)C═O or one or more of X″ is a [CON] group

linked through the amine to the ring structure of [MULTICON]

Where CL is

m in CL is an integer from 0 to 12, often 0, 1, 2, 3, 4, 5 or 6;and iL is 0 or 1, often 1;the substitutent RCON is H or a C₁-C₃ alkyl, preferably H or CH₃ andn″ is 0, 1, 2 or 3;and Z and Y are each independently

or a pharmaceutically acceptable salt, stereoisomer, solvate orpolymorph thereof.

In an additional aspect of the invention, a pharmaceutical compositioncomprises an effective amount of a bifunctional/multifunctional compoundas described above, optionally and preferably in combination with apharmaceutically acceptable carrier, additive or excipient. Inalternative aspects, pharmaceutical combination compositions comprise aneffective amount of at least one bifunctional/multifunctional compoundas described herein, in combination with at least one additional agentwhich is used to treat cancer, including prostate cancer, especiallyincluding metastatic prostate cancer or a secondary condition or effectof cancer, especially prostate cancer (e.g., bone pain, hyperplasia,osteoporosis, etc. as otherwise described herein).

In a further aspect of the invention, compounds according to the presentinvention are used to treat or reduce the likelihood of cancer,including metastatic cancer in a patient, especially prostate cancer inmale patients in need thereof and to reduce the likelihood that acancer, especially prostate cancer, will metastasize or that a cancer inremission will recur. The method of treating cancer comprisesadministering to a patient in need an effective amount of atrifunctional chimeric compound as otherwise described herein incombination with a pharmaceutically acceptable carrier, additive orexcipient, optionally in further combination with at least oneadditional agent which is effective in treating cancer, especiallyincluding prostate cancer, metastatic cancer or one or more of itssecondary conditions or effects.

The present invention also relates to a method for inhibiting prostatecancer to reduce or inhibit the spread or metastasis of the cancer intoother tissues of the patients' body, especially including bones, thelymph (lymph nodes) system, bladder, vas deferens, kidneys, liver, lungsand brain, among others.

The present invention also relates to instances in which destruction ofnon-cancerous cells which possess PSMA can be of therapeutic use,especially in cancer therapy. For example, given that PSMA is found onthe neovasculare of many non-prostatic cancer cells, but not on normalvasculature, the invention could be used for antiangiogenic therapy forother forms of cancer by targeting the neovasculature of those cancersand inhibiting the growth and spread of the cancer.

DETAILED DESCRIPTION OF THE INVENTION

The following terms are used to describe the present invention. Ininstances where a term is not specifically defined herein, that term isgiven an art-recognized meaning by those of ordinary skill applying thatterm in context to its use in describing the present invention.

The term “compound”, as used herein, unless otherwise indicated, refersto any specific chemical compound, preferably SyAMs disclosed herein andincludes tautomers, regioisomers, geometric isomers, and whereapplicable, optical isomers (enantiomers) thereof, as well aspharmaceutically acceptable salts and derivatives (including prodrugforms) thereof. Within its use in context, the term compound generallyrefers to a single compound, but also may include other compounds suchas stereoisomers, regioisomers and/or optical isomers (including racemicmixtures) as well as specific enantiomers or enantiomerically enrichedmixtures of disclosed compounds. The term also refers, in context toprodrug forms of compounds which have been modified to facilitate theadministration and delivery of compounds to a site of activity. It isnoted that in describing the present compounds, numerous substituents,linkers and connector molecules and variables associated with same,among others, are described. It is understood by those of ordinary skillthat molecules which are described herein are stable compounds asgenerally described hereunder.

The term “patient” or “subject” is used throughout the specificationwithin context to describe an animal, generally a mammal and preferablya human, to whom treatment, including prophylactic treatment(prophylaxis), with the compositions according to the present inventionis provided. For treatment of those infections, conditions or diseasestates which are specific for a specific animal such as a human patientor a patient of a particular gender, such as a human male patient, theterm patient refers to that specific animal. Compounds according to thepresent invention are useful for the treatment of cancer, especiallyincluding prostate cancer and in particular, metastatic prostate cancer.

The term “effective” is used herein, unless otherwise indicated, todescribe an amount of a compound or composition (one or more SyAMs aloneor in combination) which, in context, is used to produce or effect anintended result, whether that result relates to the inhibition of thecancer or the treatment of a subject for secondary conditions, diseasestates or manifestations of cancer as otherwise described herein. Thisterm subsumes all other effective amount or effective concentrationterms (including the term “therapeutically effective”) which areotherwise described in the present application.

The terms “treat”, “treating”, and “treatment”, etc., as used herein,refer to any action providing a benefit to a patient at risk for cancer,especially prostate cancer or metastasis of prostate cancer, includingimprovement in the condition through lessening or suppression of atleast one symptom, inhibition of cancer growth, reduction in cancercells or tissue, prevention or delay in progression of metastasis of thecancer, prevention or delay in the onset of disease states or conditionswhich occur secondary to cancer or remission or cure of the cancer,among others. Treatment, as used herein, encompasses both prophylacticand therapeutic treatment. The term “prophylactic” when used, means toreduce the likelihood of an occurrence or the severity of an occurrencewithin the context of the treatment of cancer, including cancermetastasis as otherwise described hereinabove.

The term “neoplasia” refers to the uncontrolled and progressivemultiplication of tumor cells, under conditions that would not elicit,or would cause cessation of, multiplication of normal cells. Neoplasiaresults in a “neoplasm”, which is defined herein to mean any new andabnormal growth, particularly a new growth of tissue, in which thegrowth of cells is uncontrolled and progressive. Thus, neoplasiaincludes “cancer”, which herein refers to a proliferation of tumor cellshaving the unique trait of loss of normal controls, resulting inunregulated growth, lack of differentiation, local tissue invasion,and/or metastasis.

As used herein, neoplasms include, without limitation, morphologicalirregularities in cells in tissue of a subject or host, as well aspathologic proliferation of cells in tissue of a subject, as comparedwith normal proliferation in the same type of tissue. Additionally,neoplasms include benign tumors and malignant tumors (e.g., colontumors) that are either invasive or noninvasive. Malignant neoplasms aredistinguished from benign neoplasms in that the former show a greaterdegree of anaplasia, or loss of differentiation and orientation ofcells, and have the properties of invasion and metastasis. Examples ofneoplasms or neoplasias from which the target cell of the presentinvention may be derived include, without limitation, carcinomas (e.g.,squamous-cell carcinomas, adenocarcinomas, hepatocellular carcinomas,and renal cell carcinomas), particularly those of the bladder, bowel,breast, cervix, colon, esophagus, head, kidney, liver, lung, neck,ovary, pancreas, prostate, and stomach; leukemias; benign and malignantlymphomas, particularly Burkitt's lymphoma and Non-Hodgkin's lymphoma;benign and malignant melanomas; myeloproliferative diseases; sarcomas,particularly Ewing's sarcoma, hemangiosarcoma, Kaposi's sarcoma,liposarcoma, myosarcomas, peripheral neuroepithelioma, and synovialsarcoma; tumors of the central nervous system (e.g., gliomas,astrocytomas, oligodendrogliomas, ependymomas, gliobastomas,neuroblastomas, ganglioneuromas, gangliogliomas, medulloblastomas,pineal cell tumors, meningiomas, meningeal sarcomas, neurofibromas, andSchwannomas); germ-line tumors (e.g., bowel cancer, breast cancer,prostate cancer, cervical cancer, uterine cancer, lung cancer, ovariancancer, testicular cancer, thyroid cancer, astrocytoma, esophagealcancer, pancreatic cancer, stomach cancer, liver cancer, colon cancer,and melanoma); mixed types of neoplasias, particularly carcinosarcomaand Hodgkin's disease; and tumors of mixed origin, such as Wilms' tumorand teratocarcinomas, which may be treated by one or more compoundsaccording to the present invention. See, (Beers and Berkow (eds.), TheMerck Manual of Diagnosis and Therapy, 17.sup.th ed. (WhitehouseStation, N.J.: Merck Research Laboratories, 1999) 973-74, 976, 986, 988,991.

Because of the activity of the present compounds as anti-angiogeniccompounds, the present invention has general applicability treatingvirtually any cancer in any tissue, thus the compounds, compositions andmethods of the present invention are generally applicable to thetreatment of cancer. Given that the protein target is found on theneovasculature of most non-prostatic cancer cells, the compounds in thepresent invention may also serve as an antiangiogenic therapy for othercancer types. Most often, the compounds are used to treat prostatecancer, and metastatic prostate cancer, as well as reducing thelikelihood that prostate cancer will not metastasize.

In certain particular aspects of the present invention, the cancer whichis treated is prostate cancer or metastatic prostate cancer. Separately,metastatic prostate cancer may be found in virtually all tissues of acancer patient in late stages of the disease, typically metastaticprostate cancer is found in seminal vesicles, lymph system/nodes(lymphoma), in bones, in bladder tissue, in kidney tissue, liver tissueand in virtually any tissue, including brain (brain cancer/tumor). Thus,the present invention is generally applicable and may be used to treatany cancer in any tissue, regardless of etiology.

The term “tumor” is used to describe a malignant or benign growth ortumefacent.

The term “prostate cancer” is used to describe a disease in which cancerdevelops in the prostate, a gland in the male reproductive system. Itoccurs when cells of the prostate mutate and begin to multiplyuncontrollably. These cells may metastasize (metastatic prostate cancer)from the prostate to virtually any other part of the body, particularlythe bones and lymph nodes, but the kidney, bladder and even the brain,among other tissues. Prostate cancer may cause pain, difficulty inurinating, problems during sexual intercourse, erectile dysfunction.Other symptoms can potentially develop during later stages of thedisease.

Rates of detection of prostate cancers vary widely across the world,with South and East Asia detecting less frequently than in Europe, andespecially the United States. Prostate cancer develops most frequentlyin men over the age of fifty and is one of the most prevalent types ofcancer in men. However, many men who develop prostate cancer never havesymptoms, undergo no therapy, and eventually die of other causes. Thisis because cancer of the prostate is, in most cases, slow-growing, andbecause most of those affected are over the age of 60. Hence, they oftendie of causes unrelated to the prostate cancer. Many factors, includinggenetics and diet, have been implicated in the development of prostatecancer. The presence of prostate cancer may be indicated by symptoms,physical examination, prostate specific antigen (PSA), or biopsy. Thereis concern about the accuracy of the PSA test and its usefulness inscreening. Suspected prostate cancer is typically confirmed by taking abiopsy of the prostate and examining it under a microscope. Furthertests, such as CT scans and bone scans, may be performed to determinewhether prostate cancer has spread.

Treatment options for prostate cancer with intent to cure are primarilysurgery and radiation therapy. Other treatments such as hormonaltherapy, chemotherapy, proton therapy, cryosurgery, high intensityfocused ultrasound (HIFU) also exist depending on the clinical scenarioand desired outcome. The present invention may be used to enhance anyone or more of these therapies or to supplant them.

The age and underlying health of the man, the extent of metastasis,appearance under the microscope, and response of the cancer to initialtreatment are important in determining the outcome of the disease. Thedecision whether or not to treat localized prostate cancer (a tumor thatis contained within the prostate) with curative intent is a patienttrade-off between the expected beneficial and harmful effects in termsof patient survival and quality of life.

An important part of evaluating prostate cancer is determining thestage, or how far the cancer has spread. Knowing the stage helps defineprognosis and is useful when selecting therapies. The most common systemis the four-stage TNM system (abbreviated from Tumor/Nodes/Metastases).Its components include the size of the tumor, the number of involvedlymph nodes, and the presence of any other metastases.

The most important distinction made by any staging system is whether ornot the cancer is still confined to the prostate or is metastatic. Inthe TNM system, clinical T1 and T2 cancers are found only in theprostate, while T3 and T4 cancers have spread elsewhere and metastasizedinto other tissue. Several tests can be used to look for evidence ofspread. These include computed tomography to evaluate spread within thepelvis, bone scans to look for spread to the bones, and endorectal coilmagnetic resonance imaging to closely evaluate the prostatic capsule andthe seminal vesicles. Bone scans often reveal osteoblastic appearancedue to increased bone density in the areas of bone metastasis—oppositeto what is found in many other cancers that metastasize. Computedtomography (CT) and magnetic resonance imaging (MRI) currently do notadd any significant information in the assessment of possible lymph nodemetastases in patients with prostate cancer according to ameta-analysis.

Prostate cancer is relatively easy to treat if found early. After aprostate biopsy, a pathologist looks at the samples under a microscope.If cancer is present, the pathologist reports the grade of the tumor.The grade tells how much the tumor tissue differs from normal prostatetissue and suggests how fast the tumor is likely to grow. The Gleasonsystem is used to grade prostate tumors from 2 to 10, where a Gleasonscore of 10 indicates the most abnormalities. The pathologist assigns anumber from 1 to 5 for the most common pattern observed under themicroscope, then does the same for the second most common pattern. Thesum of these two numbers is the Gleason score. The Whitmore-Jewett stageis another method sometimes used. Proper grading of the tumor iscritical, since the grade of the tumor is one of the major factors usedto determine the treatment recommendation.

Early prostate cancer usually causes no symptoms. Often it is diagnosedduring the workup for an elevated PSA noticed during a routine checkup.Sometimes, however, prostate cancer does cause symptoms, often similarto those of diseases such as benign prostatic hypertrophy. These includefrequent urination, increased urination at night, difficulty startingand maintaining a steady stream of urine, blood in the urine, andpainful urination. Prostate cancer is associated with urinarydysfunction as the prostate gland surrounds the prostatic urethra.Changes within the gland therefore directly affect urinary function.Because the vas deferens deposits seminal fluid into the prostaticurethra, and secretions from the prostate gland itself are included insemen content, prostate cancer may also cause problems with sexualfunction and performance, such as difficulty achieving erection orpainful ejaculation.

Advanced prostate cancer can spread to other parts of the body and thismay cause additional symptoms. The most common symptom is bone pain,often in the vertebrae (bones of the spine), pelvis or ribs. Spread ofcancer into other bones such as the femur is usually to the proximalpart of the bone. Prostate cancer in the spine can also compress thespinal cord, causing leg weakness and urinary and fecal incontinence.

The specific causes of prostate cancer remain unknown. A man's risk ofdeveloping prostate cancer is related to his age, genetics, race, diet,lifestyle, medications, and other factors. The primary risk factor isage. Prostate cancer is uncommon in men less than 45, but becomes morecommon with advancing age. The average age at the time of diagnosis is70. However, many men never know they have prostate cancer.

A man's genetic background contributes to his risk of developingprostate cancer. This is suggested by an increased incidence of prostatecancer found in certain racial groups, in identical twins of men withprostate cancer, and in men with certain genes. Men who have a brotheror father with prostate cancer have twice the usual risk of developingprostate cancer. Studies of twins in Scandinavia suggest that fortypercent of prostate cancer risk can be explained by inherited factors.However, no single gene is responsible for prostate cancer; manydifferent genes have been implicated. Two genes (BRCA1 and BRCA2) thatare important risk factors for ovarian cancer and breast cancer in womenhave also been implicated in prostate cancer.

Dietary amounts of certain foods, vitamins, and minerals can contributeto prostate cancer risk. Dietary factors that may increase prostatecancer risk include low intake of vitamin E, the mineral selenium, greentea and vitamin D. A large study has implicated dairy, specificallylow-fat milk and other dairy products to which vitamin A palmitate hasbeen added. This form of synthetic vitamin A has been linked to prostatecancer because it reacts with zinc and protein to form an unabsorbablecomplex. Prostate cancer has also been linked to the inclusion of bovinesomatotropin hormone in certain dairy products.

There are also some links between prostate cancer and medications,medical procedures, and medical conditions. Daily use ofanti-inflammatory medicines such as aspirin, ibuprofen, or naproxen maydecrease prostate cancer risk. Use of the cholesterol-lowering drugsknown as the statins may also decrease prostate cancer risk. Infectionor inflammation of the prostate (prostatitis) may increase the chancefor prostate cancer, and infection with the sexually transmittedinfections chlamydia, gonorrhea, or syphilis seems to increase risk.Obesity and elevated blood levels of testosterone may increase the riskfor prostate cancer.

Prostate cancer is classified as an adenocarcinoma, or glandular cancer,that begins when normal semen-secreting prostate gland cells mutate intocancer cells. The region of prostate gland where the adenocarcinoma ismost common is the peripheral zone. Initially, small clumps of cancercells remain confined to otherwise normal prostate glands, a conditionknown as carcinoma in situ or prostatic intraepithelial neoplasia (PIN).Although there is no proof that PIN is a cancer precursor, it is closelyassociated with cancer. Over time these cancer cells begin to multiplyand spread to the surrounding prostate tissue (the stroma) forming atumor. Eventually, the tumor may grow large enough to invade nearbyorgans such as the seminal vesicles or the rectum, or the tumor cellsmay develop the ability to travel in the bloodstream and lymphaticsystem. Prostate cancer is considered a malignant tumor because it is amass of cells which can invade other parts of the body. This invasion ofother organs is called metastasis. Prostate cancer most commonlymetastasizes to the bones, lymph nodes, rectum, and bladder.

In prostate cancer, the regular glands of the normal prostate arereplaced by irregular glands and clumps of cells. When a man hassymptoms of prostate cancer, or a screening test indicates an increasedrisk for cancer, more invasive evaluation is offered. The only testwhich can fully confirm the diagnosis of prostate cancer is a biopsy,the removal of small pieces of the prostate for microscopic examination.However, prior to a biopsy, several other tools may be used to gathermore information about the prostate and the urinary tract. Cystoscopyshows the urinary tract from inside the bladder, using a thin, flexiblecamera tube inserted down the urethra. Transrectal ultrasonographycreates a picture of the prostate using sound waves from a probe in therectum.

After biopsy, the tissue samples are then examined under a microscope todetermine whether cancer cells are present, and to evaluate themicroscopic features (or Gleason score) of any cancer found. Inaddition, tissue samples may be stained for the presence of PSA andother tumor markers in order to determine the origin of malignant cellsthat have metastasized. A number of other potential approaches fordiagnosis of prostate cancer are ongoing such as early prostate cancerantigen-2 (EPCA-2), and prostasome analysis.

In addition to therapy using the compounds according to the presentinvention, therapy (including prophylactic therapy) for prostate cancersupports roles in reducing prostate cancer for dietary selenium, vitaminE, lycopene, soy foods, vitamin D, green tea, omega-3 fatty acids andphytoestrogens. The selective estrogen receptor modulator drugtoremifene has shown promise in early trials. Two medications whichblock the conversion of testosterone to dihydrotestosterone (and reducethe tendency toward cell growth), finasteride and dutasteride, are shownto be useful. The phytochemicals indole-3-carbinol and diindolylmethane,found in cruciferous vegetables (califlower and broccholi), havefavorable antiandrogenic and immune modulating properties. Prostatecancer risk is decreased in a vegetarian diet.

Treatment for prostate cancer may involve active surveillance, surgery(prostatecomy or orchiectomy), radiation therapy including brachytherapy(prostate brachytherapy) and external beam radiation as well as hormonaltherapy. There are several forms of hormonal therapy which include thefollowing, each of which may be combined with compounds according to thepresent invention.

-   -   Antiandrogens such as flutamide, bicalutamide, nilutamide, and        cyproterone acetate which directly block the actions of        testosterone and DHT within prostate cancer cells.    -   Medications such as ketoconazole and aminoglutethimide which        block the production of adrenal androgens such as DHEA. These        medications are generally used only in combination with other        methods that can block the 95% of androgens made by the        testicles. These combined methods are called total androgen        blockade (TAB), which can also be achieved using antiandrogens.    -   GnRH modulators, including agonists and antagonists. GnRH        antagonists suppress the production of LH directly, while GnRH        agonists suppress LH through the process of downregulation after        an initial stimulation effect. Abarelix is an example of a GnRH        antagonist, while the GnRH agonists include leuprolide,        goserelin, triptorelin, and buserelin.    -   The use of abiraterone acetate can be used to reduce PSA levels        and tumor sizes in aggressive end-stage prostate cancer for as        high as 70% of patients. Sorafenib may also be used to treat        metastatic prostate cancer.

Each treatment described above has disadvantages which limit its use incertain circumstances. GnRH agonists eventually cause the same sideeffects as orchiectomy but may cause worse symptoms at the beginning oftreatment. When GnRH agonists are first used, testosterone surges canlead to increased bone pain from metastatic cancer, so antiandrogens orabarelix are often added to blunt these side effects. Estrogens are notcommonly used because they increase the risk for cardiovascular diseaseand blood clots. The antiandrogens do not generally cause impotence andusually cause less loss of bone and muscle mass. Ketoconazole can causeliver damage with prolonged use, and aminoglutethimide can cause skinrashes.

Palliative care for advanced stage prostate cancer focuses on extendinglife and relieving the symptoms of metastatic disease. As noted above,abiraterone acetate shows some promise in treating advance stageprostate cancer as does sorafenib. Chemotherapy may be offered to slowdisease progression and postpone symptoms. The most commonly usedregimen combines the chemotherapeutic drug docetaxel with acorticosteroid such as prednisone. Bisphosphonates such as zoledronicacid have been shown to delay skeletal complications such as fracturesor the need for radiation therapy in patients with hormone-refractorymetastatic prostate cancer. Alpharadin may be used to target bonemetastasis. The phase II testing shows prolonged patient survival times,reduced pain and improved quality of life.

Bone pain due to metastatic disease is treated with opioid painrelievers such as morphine and oxycodone. External beam radiationtherapy directed at bone metastases may provide pain relief. Injectionsof certain radioisotopes, such as strontium-89, phosphorus-32, orsamarium-153, also target bone metastases and may help relieve pain.

As an alternative to active surveillance or definitive treatments,alternative therapies may also be used for the management of prostatecancer. PSA has been shown to be lowered in men with apparent localizedprostate cancer using a vegan diet (fish allowed), regular exercise, andstress reduction. Many other single agents have been shown to reducePSA, slow PSA doubling times, or have similar effects on secondarymarkers in men with localized cancer in short term trials, such aspomegranate juice or genistein, an isoflavone found in various legumes.

Manifestations or secondary conditions or effects of metastatic andadvanced prostate cancer may include anemia, bone marrow suppression,weight loss, pathologic fractures, spinal cord compression, pain,hematuria, ureteral and/or bladder outlet obstruction, urinaryretention, chronic renal failure, urinary incontinence, and symptomsrelated to bony or soft-tissue metastases, among others.

Additional prostate drugs which can be used in combination with thechimeric antibody recruiting compounds according to the presentinvention include, for example, the enlarged prostate drugs/agents, aswell as eulexin, flutamide, goserelin, leuprolide, lupron, nilandron,nilutamide, zoladex and mixtures thereof. Enlarged prostate drugs/agentsas above, include for example, ambenyl, ambophen, amgenal, atrosept,bromanyl, bromodiphenhydramine-codeine, bromotuss-codeine, cardura,chlorpheniramine-hydrocodone, ciclopirox, clotrimazole-betamethasone,dolsed, dutasteride, finasteride, flomax, gecil, hexalol, lamisil,lanased, loprox, lotrisone, methenamine, methen-bella-meth Bl-phen sal,meth-hyos-atrp-M blue-BA-phsal, MHP-A, mybanil, prosed/DS, Ro-Sed, S-TForte, tamsulosin, terbinafine, trac, tussionex, ty-methate, uramine,uratin, uretron, uridon, uro-ves, urstat, usept and mixtures thereof.

The term “Immune binding terminal moiety”, “Immune binding terminus”, or[IBT] is use to described that portion of a chimeric compound accordingto the present invention which comprises a molecule which binds toFcγRI.receptor on an immune effector cell (macrophage, neutrophil,eosinophil, dendritic cell, etc.) and facilitates opsonization,phagocytosis, cytotoxicity and the death of cancer cells. A preferredFcRI binding moiety for use in the present invention is CP33, presentedin attached FIG. 2.

Representative [IBT] groups for use in the present invention includethose which appear in attached FIG. 14. Each of these compounds can beused to modulate FcγRI receptors which interact with immune effectorcells (macrophages, eosinophils, neutrophils) to induce phagocytic andother activity by these effector cells against cancer cells as otherwisedescribed herein. In addition, these compounds may also provide anability to induce a long-term immune response to cancer cells in apatient, thus reducing the likelihood of a recurrence after cancerremission.

Representative [IBT] groups include for example, CP33 (a preferred IBT),YU158145 (4861-0063), YU160469 (7165-0402), YU160642 (7527-0236),YU160645 (7527-0311), YU160647 (7527-0320), YU164313 (D086-0504),YU164340 (D089-0267), YU164343 (D089-0287), YU164367 (D089-0353),YU163512 (C766-0140), YU163538 (C766-0571), YU163540 (C766-0578),YU163511 (C766-0135), YU163552 (C798-0145), YU163554 (C798-0169),YU160794 (7889-2431), YU163387 (C720-0562), YU163388 (C720-0563),YU162384 (C218-0192), YU162389 (C218-0271), YU162392 (C218-0284),YU162393 (C218-0288), YU162402 (C218-0460), YU162408 (C218-0524),YU165568 (D420-6099), P680-0123 (YU170286:01), YU165561 (D420-5349),YU165531 (D420-4922), YU165550 (D420-5220), YU169577 (M050-0297),YU162636 (C291-0121), YU163100 (C611-0416), YU163102 (C611-0421),YU162257 (C200-0357), YU169182 (K891-0143), YU169197 (K891-0201),YU162415 (C218-0870), YU162417 (C218-0879), YU162413 (C218-0864),YU162414 (C218-0868) and YU162730 (C301-9341), which are modified at afree hydroxyl or free amine to link to other components of the compoundsaccording to the present invention. Each of these immune modulatingcompounds is presented as a chemical structure in FIG. 14 withattachment indicated at a free hydroxyl or free amine of the compound.Attachment on each of these molecules to a linker and the rest of themolecules according to the present invention is on a free hydroxyl or afree amine, as represented in FIG. 14 (bond intersecting another bond atO or N in each compound, accordingly). The [IBT] most often used in thepresent invention is CP33, because it has shown the greater activity asa modulator of FcγRI. CP33 is also represented in FIG. 14.

The term “cell binding terminal moiety”, “cell binding terminus” or“cell binding moiety” is use to described that portion of a chimericcompound according to the present invention which comprises at least onesmall molecule or moiety which can bind specifically to prostatespecific membrane antigen (PSMA).

Preferred CBT groups for use in the present invention are set forthbelow:

Where X₁ and X₂ are each independently CH₂, O, NH or S;X₃ is O, CH₂, Me, S(O), S(O)₂, —S(O)₂O, —OS(O)₂, or OS(O)₂O;R¹ is H, a C₁-C₃ alkyl group, or a —C(O)(C₁-C₃) group;k is an integer from 0 to 20, 8 to 12, 1 to 15, 1 to 10, 1 to 8, 1 to 6,1, 2, 3, 4, 5 or 6;or a salt or enantiomer thereof.

A preferred CBT group for use in the present invention is the group

Where k is 2, 3 or 4, preferably 3. This CBT group, as well as theothers, optionally has an amine group or other functional group at thedistill end of the alkylene group (k) such that k is formed from, forexample, a lysine amino acid, such that the amine group or otherfunctional group may participate in further reactions to form a linker,a triazole or other difunctional connector group [CON] or amultifunctional group [MULTICON] as otherwise described herein.

The term “linker” refers to a chemical entity which connects the cellbinding terminus moiety [CBT] or immune (Fc receptor) binding terminus[IBT] to the difunctional connector moiety moiety/molecule [CON] and/orthe multifunctional connector moiety/molecule [MULTICON]. It is notedthat each linker may be connected to [MULTICON} through an optionaldifunctional connector molecule [CON} as otherwise disclosed here. Thelinker is non-labile and also may be comprised of one or more linkergroups to provide extended linkers as otherwise disclosed herein. Eachlinker may be directly linked to a [CBT] or an [IBT] group especiallybetween the [ABT] moiety and the multifunctional connector molecule[MULTICON} and the [CBT] moiety and the multifunctional connectormolecule [MULTICON]. In certain instances, the CBT group may be linkeddirectly to a difunctional connector group [CON] which is further linkedto a linker group and optionally, a [MULTICON] group. The linker betweenthe two active functional portions of the molecule, i.e., the immunebinding terminus [IBT] and the cell binding terminus [CBT] ranges fromabout 5 Å to about 50 Å or more in length, about 6 Å to about 45 Å inlength, about 7 Å to about 40 Å in length, about 8 Å to about 35 Å inlength, about 9 Å to about 30 Å in length, about 10 Å to about 25 Å inlength, about 7 Å to about 20 Å in length, about 5 Å to about 16 Å inlength, about 5 Å to about 15 Å in length, about 6 Å to about 14 Å inlength, about 10 Å to about 20 Å in length, about 1 IA to about 25 Å inlength, etc. Linkers which are based upon unnatural amino acids (i.e.,having an alkylene group with between 3 and 6, preferably 4 or 5methylene groups between the amine and acid of the amino acid monomericunit) as otherwise described herein often used. By having a linker witha length as otherwise disclosed herein, the one or more [IBT] moietiesand the one or more [CBT] moieties may be situated to take advantage ofthe biological activity of compounds according to the present inventionwhich bind to prostate specific membrane antigen (PSMA) through the[CBT] group and modulate Fc receptors, in particular, FcγRI receptors onimmune effector molecules through the [IBT] group functioningsynergistically with the [CBT] group unit to improve the compound'sability to modulate tumor lysis (e.g. phagocytosis) by immune effectorcells and better stimulate and promote immunologic memory to which thecompounds are bound. This results in the selective and targeted(synergistic) cell death of cancer cells, including metastatic cancercells, in particular, prostate cancer cells and metastatic prostratecancer cells, in whatever tissues they may reside. The selection of alinker component is based on its documented properties ofbiocompatibility, solubility in aqueous and organic media, and lowimmunogenicity/antigenicity.

Linkers which are based upon or include oligo amino acid units arepreferred for use in the present invention. These preferred linkers arebetween 2 and 100 amino acid units in length, but those which arebetween 2 and 14 amino acid units or 4 to 8 amino acid units in lengthmay be preferred. In preferred aspects, each amino acid unit is anunnatural amino acid unit as otherwise described herein, preferablyhaving between 3 and 6 methylene group in each amino acid monomericunit. Each linker may be linked with the multifunctional connectormolecule [MULTICON] through one or more difunctional connector molecules[CON] as otherwise disclosed herein.

Although numerous linkers may be used as otherwise described herein, alinker (non-labile) based upon polyethyleneglycol (PEG) linkages,polypropylene glycol linkages, or polyethyleneglycol-co-polypropyleneglycol polymers (e.g., block copolymers where the polyethylene glycolportion of the block is from 1 to 12 polyethylene glycol units in lengthand said polypropylene glycol portion of the block is from 1 to 12polypropylene glycol units in length, the total number of polyethyleneglycol units, polypropylene glycol units orpolyethyleneglycol-co-polypropyleneglycol block copolymer units beingfrom 1 to 100, 1 to 75, 1 to, 1 to 15, 1 to 12, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11 or 12 or often up to about 100 units, about 1 to 100, about 1to 75, about 1 to 60, about 1 to 50, about 1 to 35, about 1 to 25, about1 to 20, about 1 to 15, 1 to 10, about 8 to 12, about 1 to 8. Of these,polyethylene (PEG) linkages are more often used.

Alternative preferred linkers may include, for example, polyprolinelinkers and/or collagen linkers as depicted below (n is about 1 to 100,about 1 to 75, about 1 to 60, about 1 to 50, about 1 to 45, about 1 to35, about 1 to 25, about 1 to 20, about 1 to 15, 2 to 10, about 4 to 12,about 5 to 10, about 4 to 6, about 1 to 8, about 1 to 6, about 1 to 5,about 1 to 4, about 1 to 3, etc.).

Additional linkers include those according to the chemical structures:

Where R_(a) is H, C₁-C₃ alkyl or alkanol or forms a cyclic ring with R³(proline) and R³ is a side chain derived from an amino acid preferablyselected from the group consisting of alanine (methyl), arginine(propyleneguanidine), asparagine (methylenecarboxyamide), aspartic acid(ethanoic acid), cysteine (thiol, reduced or oxidized di-thiol),glutamine (ethylcarboxyamide), glutamic acid (propanoic acid), glycine(H), histidine (methyleneimidazole), isoleucine (1-methylpropane),leucine (2-methylpropane), lysine (butyleneamine), methionine(ethylmethylthioether), phenylalanine (benzyl), proline (R³ forms acyclic ring with R_(a) and the adjacent nitrogen group to form apyrrolidine group), hydroxyproline, serine (methanol), threonine(ethanol, 1-hydroxyethane), tryptophan (methyleneindole), tyrosine(methylene phenol) or valine (isopropyl);m″ is an integer between 0 to 25, preferably 1 to 10, 1 to 8, 1, 2, 3,4, 5, or 6;m (within this context) is an integer from 1 to 100, 1 to 75, 1 to 60, 1to 55, 1 to 50, 1 to 45, 1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to 10, 1to 8, 1 to 6, 1, 2, 3, 4 or 5; andn (within this context) is an integer from about 1 to 100, about 1 to75, about 1 to 60, about 1 to 50, about 1 to 45, about 1 to 35, about 1to 25, about 1 to 20, about 1 to 15, 2 to 10, about 4 to 12, about 5 to10, about 4 to 6, about 1 to 8, about 1 to 6, about 1 to 5, about 1 to4, about 1 to 3, etc.).

Another linker according to the present invention comprises apolyethylene glycol linker containing from 1 to 1 to 100, 1 to 75, 1 to60, 1 to 55, 1 to 50, 1 to 45, 1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to10, 1 to 8, 1 to 6, 1, 2, 3, 4 or 5 ethylene glycol units which may befurther linked through amide groups (which include alkylene groups oneither or both sides of the amide group containing one to five methyleneunits), keto groups (which include alkylene keto groups containing oneto five methylene units), amine groups (which include alkylene aminegroups containing one to five methylene units), alkylene groups(containing from 1 to 5 methylene units), amino acids or other moietiescompatible with polyethylene glycol groups, including difunctionalconnecting groups [CON]), [CBT] groups, [IBT] groups, [MULTICON] groupsand other linker groups including other polyethylene glycol groups(often with anywhere between 1 and 12 ethylene glycol units (e.g. 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12). Still other linkers comprisepolypeptides of amino acid residues (D or L). In another embodiment, asotherwise described herein, polypeptides may comprise non-naturallyoccurring amino acids (non-naturally occurring except for glycine) ofthe non-labile linker each of which has anywhere from 1-15 or moremethylene groups separating the amino group from the acid group, oftenfrom three to six methylene groups (3, 4, 5 or 6), and from 1 to 100peptide groups in providing a linker to the moiety, preferably 1 to 12(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12). It is noted that each of thepolypeptide linkers and other linkers (including labile linkers)identified in the present application may be further linked together orwith connector molecules/moieties [CON], [MULTICON] molecules/moieties,[IBT] groups, and/or [CBT] groups through amide groups (which includealkylene groups on either or both sides of the amide group containingone to five methylene units), keto groups (which include alkylene ketogroups containing one to five methylene units on either or both sides ofthe keto group), amine groups (which include alkylene amine groupscontaining one to five methylene units on either or both sides of theamine group), urethane groups (which include alkylene groups containingone to five methylene units on either or both sides of the urethanemoiety) alkylene groups (containing from 1 to 5 methylene units), aminoacids or other moieties compatible with the linker chemistry in order tolink components of the molecules. It is noted that in the case ofpolyethylene glycol and polypeptide linkers, the use of an additionalgroup (eg, alkylene amine or other group as described above) or a secondlinker group may be useful for joining the linker to another componentof the molecule. Additionally, more than one linker group identifiedherein may be linked together to form a linker group as otherwise usedin the present compounds, consistent with the stability of the linkerchemistries. These extended linkers are often linked through [CON}connecting groups or as otherwise described herein.

Additional preferred linkers include those according to the structure:

where R_(a) is H or a C₁-C₃ alkyl, preferably CH₃, most often H;m is an integer from 1 to 12, often 1, 2, 3, 4, 5, or 6;m″ is an integer 1, 2, 3, 4, 5, or 6, often 6;t is 0, 1, 2, 3, 4, 5, or 6; andiL is 0 or 1, often 1, wherein said linker in certain instances, may bepreferably linked to a [CON] group and a [CBT] group at one end and a[MULTICON] group on the other end; or a linker according to thestructure:

Where q is an integer from 0-12, preferably 1, 2, 3, 4, 5 or 6; andq′ is 1 to 12, often 1, 2, 3, 4, 5 or 6.

The two above linkers may be linked together to provide further linkerswhich are often used in compounds according to the present invention:

Where q is an integer from 0-12, preferably 0, 1, 2, 3, 4, 5 or 6;q′ is 1 to 12, often 1, 2, 3, 4, 5 or 6;iL is 0 or 1; andR_(L) is an amino acid or an oligopeptide (which term includes adipeptide) as otherwise described herein, especially including lysine,dilysine, or glycinelysine.

Another linker according to the present invention includes a linkerbased upon succinimide according to the chemical formula:

where each X^(S) is independently S, O or N—R^(S), preferably S;R^(S) is H or C₁₋₃ alkyl, preferably H;S_(c) is CH₂, CH₂O; or CH₂CH₂O;i is 0 or 1; andm^(S) is 0, 1, 2, 3, 4, 5, or 6.Other linkers which may be used in the present invention include linkersaccording to the chemical formula:

Where Z and Z′ are each independently a bond, —(CH₂)_(i)—O,—(CH₂)_(i)—S, —(CH₂)_(i)—N—R,

wherein said Z or Z′ group is optionally bonded to another linker group,a connector [CON], a [MULTICON] group, IBT or CBT;Each R is H, or a C₁-C₃ alkyl or alkanol group;Each R² is independently H or a C₁-C₃ alkyl group;Each Y is independently a bond, O, S or N—R;Each i is independently 0 to 100, preferably 1 to 100, 1 to 75, 1 to 60,1 to 55, 1 to 50, 1 to 45, 1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to 10,1 to 8, 1 to 6, 0, 1, 2, 3, 4 or 5;

D is

a bond, or D may be

or a polypropylene glycol or polypropylene-co-polyethylene glycol linkerhaving between 1 and 100 glycol units (1 to 75, 1 to 60, 1 to 55, 1 to50, 1 to 45, 1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to 10, 1 to 8, 1 to6, 1, 2, 3, 4 or 52 and 50, 3 and 45);with the proviso that Z, Z′ and D are not each simultaneously bonds;each i is the same as above;j is 1 to 100, 1 to 75, 1 to 60, 1 to 55, 1 to 50, 1 to 45, 1 to 40, 2to 35, 3 to 30, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1, 2, 3, 4 or 5;m (within this context) is an integer from 1 to 100, 1 to 75, 1 to 60, 1to 55, 1 to 50, 1 to 45, 1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to 10, 1to 8, 1 to 6, 1, 2, 3, 4 or 5; andn (within this context) is an integer from about 1 to 100, about 1 to75, about 1 to 60, about 1 to 50, about 1 to 45, about 1 to 35, about 1to 25, about 1 to 20, about 1 to 15, 2 to 10, about 4 to 12, about 5 to10, about 4 to 6, about 1 to 8, about 1 to 6, about 1 to 5, about 1 to4, about 1 to 3, etc.).m′ is 1 to 100, 1 to 75, 1 to 60, 1 to 55, 1 to 50, 1 to 45, 1 to 40, 2to 35, 3 to 30, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1, 2, 3, 4 or 5;m″ is an integer between 0 to 25, preferably 1 to 10, 1 to 8, 1, 2, 3,4, 5, or 6;n′ is 1 to 100, 1 to 75, 1 to 60, 1 to 55, 1 to 50, 1 to 45, 1 to 40, 2to 35, 3 to 30, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1, 2, 3, 4 or 5;

X′ is O, S or N—R;

R is as described above;R_(a) is H, C₁-C₃ alkyl or alkanol or forms a cyclic ring with R³(proline); and R³ is a side chain derived from an amino acid preferablyselected from the group consisting of alanine (methyl), arginine(propyleneguanidine), asparagine (methylenecarboxyamide), aspartic acid(ethanoic acid), cysteine (thiol, reduced or oxidized di-thiol),glutamine (ethylcarboxyamide), glutamic acid (propanoic acid), glycine(H), histidine (methyleneimidazole), isoleucine (1-methylpropane),leucine (2-methylpropane), lysine (butyleneamine), methionine(ethylmethylthioether), phenylalanine (benzyl), proline (R³ forms acyclic ring with R₄ and the adjacent nitrogen group to form apyrrolidine group), hydroxyproline, serine (methanol), threonine(ethanol, 1-hydroxyethane), tryptophan (methyleneindole), tyrosine(methylene phenol) or valine (isopropyl), wherein said sidechain of saidamino acid (often lysine) is optionally linked or a pharmaceuticallyacceptable salt thereof. In certain embodiments, the amino acid may belinked through the sidechain of the amino acid. In certain embodiments,the amino acid is often lysine because of its trifunctionality whereinthe amine of the sidechain is used to link other linkers and/or othercomponents of the molecule. It is noted that an amino acid which hastrifunctionality and the sidechain is used to create the linker, theamino acid (often lysine) may be end-capped at the carboxylic acid withan amine group which is optionally substituted with a C₁-C₁₂ alkylgroup, preferably a C₁-C₃ alkyl group) or at the amine terminus with anacyl group.

It is noted that for each linker, one or more of the linking groups asdepicted herein may be extended through binding with one or moredifunctional connector group [CON], which is described in greater detailhereinbelow. For example, a polyethylene glycol linker (e.g. from 1 to12 ethylene glycol units, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12)extended to one or more polyethylene glycole linker(s) (e.g. each linkerbeing from 1 to 12 ethylene glycol units, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11 or 12) through one or more [CON[ groups as otherwise describedherein, including an amide [CON] group(s) represents additionalembodiments of the present invention.

As discussed, certain preferred linkers for use in the present inventioninclude a linker group as shown in compounds 1, 2 or 3 of FIG. 2 whichoften links a [CBT] moiety to a [MULTICON] molecule according to thechemical structure:

where R_(a) is H or CH₃, most often H;m is an integer from 1 to 12, often 1, 2, 3, 4, 5, or 6;m″ is an integer 1, 2, 3, 4, 5, or 6, often 6;t is 0, 1, 2, 3, 4, 5, or 6; andiL is 0 or 1, often 1, wherein L may be optionally linked to a [CON]group and a [CBT] group at one end and a [MULTICON] group on the otherend; or

As described above, alternatively, often L is a complex linker group (asdepicted in compound 3 of FIG. 2) which is made up of an ethylene oxidecontaining amine group

where q is 0 to 12, 1, 2, 3, 4, 5 or 6;q′ is 1 to 12, often 1, 2, 3, 4, 5 or 6,iL is 0 or 1 wherein the keto group is linked to an amino acid group orto an oligopeptide (including a dipeptide) and the amine group isoptionally linked through a diketo group (often to [IBT], more oftenCP33),

where q is the same as above, and an amino acid, often lysine, or anamino acid dipeptide, often dilysine. In preferred aspects, one of thelysines of the dipeptide is directly bonded to CP33, and another aminoacid (often lysine) links the above ethylene oxide amine group to a[MULTICON] group through a [CON] group, most often a triazole. Thechemical structure of the complex linker described above may berepresented by the chemical structure:

Where q is an integer from 0-12, preferably 0, 1, 2, 3, 4, 5 or 6;q′ is 1 to 12, often 1, 2, 3, 4, 5 or 6;iL is 0 or 1; andR_(L) is an amino acid or an oligopeptide (which term includes adipeptide) as otherwise described herein, especially including lysine,dilysine, or glycinelysine.

In certain additional embodiments, the linker group L is an amino acid,a dipeptide or an oligopeptide containing from 1 to 12, preferably 1 to6 amino acid monomers or more. In certain embodiments, the oligopeptideis a dipeptide and the dipeptide is a dilysine or a glycinelysinedipeptide. When lysine is used as an amino acid in an oligopeptidelinker, the sidechain alkylene amine is often used to link other linkergroups or other components in the molecule.

The term “multifunctional connector”, symbolized by [MULTICON], is usedto describe a chemical group or molecule which is optionally included inchimeric compounds according to the present invention which forms fromthe reaction product of an activated IBT-linker with a CBT moiety (whichalso is preferably activated) or an IBT moiety with an activatedlinker-CBT as otherwise described herein. Numerous other syntheticapproaches are possible. The synthetic chemistry used to synthesizecompounds according to the present invention is presented in detailherein. The connector group is the resulting moiety which forms from thefacile condensation of at least three separate chemical fragments whichcontain reactive groups which can provide connector groups as otherwisedescribed to produce chimeric compounds according to the presentinvention. It is noted that a multifunctional connector moiety ormolecule [MULTICON] is readily distinguishable from a linker in that themultifunctional connector is the result of a specific chemistry which isused provide chimeric compounds according to the present invention.

Connecting moieties in the present invention include at least onemultifunctional moiety or molecule [MULTICON] which contains three ormore functional groups which may be used to covalently bind (preferably,through a linker) to at least one [IBT] group and at least one [CBT]group, thus linking each of these functional groups into a singlecompound. Multifunctional connector groups for use in the presentinvention include moities which have at least three or more functionalgroups which can bind to linkers to which are bound [IBT] and [CBT]groups in order to provide compounds which contain at least one andpreferably more than one [IBT] and [CBT] pursuant to the presentinvention. These multifunctional connector moieties may also bind toother multifunctional connector molecules in order to create compoundscontaining a number of [IBT] and [CBT] groups as defined herein.

Multifunctional connector molecules [MULTICON] comprise any molecule ormoiety which contains at least three groups which may be linked to [IBT]and [CBT] groups, linkers and/or other connector groups (includingdifunctional and multifunctional connector groups) and preferablycomprise five or six-membered aryl or heteroaryl groups (especiallysix-membered ring groups) exemplified by multifunctional, especiallytrifunctional or tetrafunctional aryl or heteroaryl groups, includingphenyl, pyridyl, pyrimidinyl, 1,3,5-triazinyl, 1,2,3-triazinyl,1,2,4-triazinyl groups, each of which is substituted with at least 3 andup to 6 functional groups or other groups as defined herein. Thesefunctional groups may be derived from nucleophilic or electrophilicgroups on the multifunctional connector molecule precursor (themultifunctional connector molecule which forms the [MULTICON] moiety infinal compounds according to the present Mention) which are condensedonto linker groups (containing, for example an [IBT] group and a [CBT]group which contain a group which can be linked to the [MULTICON]moiety. [MULTICON] groups which are used in the present inventionpreferably include substituted phenyl, pyridyl, pyrimidinyl and1,3,5-triazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl groups, especiallygroups according to the chemical structure:

where Y₄ is C—H or N; andEach X″ is independently derived from an electrophilic or nucleophilicgroup, preferably (CH₂)_(n″)O, (CH₂)_(n″)N^(RCON), (CH₂)_(n″)S,(CH₂)_(n″) or (CH₂)_(n″)C═O or when said [MULTICON] group is a ringstructure, X″ is optionallya [CON] group, often a triazole group, linked to the ring structure,often directly to the ring structure;the substitutent RCON is H or a C₁-C₃ alkyl, preferably H or CH₃ andn″ is 0, 1, 2 or 3, andr is an integer from 1-12, often, 1, 2, 3, 4, 5, or 6.

The term “difunctional connector group” or [CON] is used to describe adifunctional group which connects two (or more) portions of a linkergroup to extend the length of the linker group. In certain embodiments,a linker group is reacted with or forms a [CON] group with anotherlinker group to form an extended linker group. The reaction product ofthese groups results in an identifiable connector group [CON] which isdistinguishable from the linker group as otherwise described herein. Itis further noted that there may be some overlap between the descriptionof the difunctional connector group and the linker group, especiallywith respect to more common connector groups such as amide groups,oxygen (ether), sulfur (thioether) or amine linkages, urea or carbonate—OC(O)O— groups as otherwise described herein. It is noted that adifunctional connector molecule [CON] used hereunder is often connectedto two parts of a linker group which binds [IBT] and [CBT] to themultifunctional connector molecule [MULTICON]. Alternatively, a [CON]group may be directly linked to a [IBT] group or more often, a [CBT]group, as well as a [MULTICON] group as described herein.

Common difunctional connector groups [CON] which are used in the presentinvention, principally to link one end of a linker to another end of alinker to provide a longer linker include the following chemical groups:

Where X² is O, S, NR⁴, S(O), S(O)₂, —S(O)₂O, —OS(O)₂, or OS(O)₂O;X³ is O, S, NR⁴; andR⁴ is H, a C₁-C₃ alkyl or alkanol group, or a —C(O)(C₁-C₃) group.

In embodiments, [CON] is often a

group;where CL is

m in CL is an integer from 0 to 12, often 0, 1, 2, 3, 4, 5 or 6;and iL is 0 or 1, often 1;In certain embodiments, this [CON] group is often linked through theamine of the triazole to the ring structure of [MULTICON].

As discussed hereinabove, it is noted that each of the [ABT] and [CBT]functional groups may be further linked to a chemical moiety which bondstwo or more of the above connector/multiconnector groups into a largermultifunctional connector, thus providing complex multifunctionalcompounds comprising more than one [IBT] and [CBT] groups within themultifunctional compound.

The term “end-cap” or “end-capping” is used to describe a non-reactivechemical moiety which is bonded to a free carboxylic acid group or afree amine group in an amino acid which is often used as a linker incompounds according to the present invention. Preferred end-cappinggroups for carboxylic acids in the present invention are amine groupswhich are optionally substituted with a C₁-C₁₂ alkyl group, preferably aC₁-C₃ alkyl group. By forming an amide with the active carboxylic acid,the carboxylic acid becomes a stable non-reactive moiety in the presentcompounds. In the case of amine groups, these are end-capped preferablywith acyl groups or urethane groups, preferably acyl groups to formstable, unreactive amides.

The term “acyl” is used to describe a group according to the chemicalstructure which often contains a C₁ to C₂₀, often a C₂ to C₂₀, linear,branched or cyclic alkyl chain linked to a keto group, thus forming anunreactive amide with a free amine in compounds according to the presentinvention. The acyl group on the free amine (often of an amino acid usedin linkers in compounds according to the present invention results in anstable, unreactive amide (although the amide may be cleave afteradministration of compounds according to the present invention, thusproviding, in certain instances, prodrug embodiments of compoundsaccording to the present invention. Acyl groups according to the presentinvention may be represented by the structure:

where R₄ is a C₁ to C₂₀ linear, branched or cyclic alkyl group,alkoxyalkyl, aryloxyalkyl, such as phenoxymethyl, aryl, alkoxy, amongothers. Preferred acyl groups are those where R₄ is a C₁ to C₁₀ alkylgroup. Acyl groups according to the present invention also include, forexample, those acyl groups derived from benzoic acid and related acids,such as substituted benzoic acid, succinic, capric and caproic, lauric,myristic, palmitic, stearic and oleic groups, among numerous others. Oneof ordinary skill in the art will recognize the acyl groups which willhave utility in the present invention, either to end-cap reactive aminegroups to produce unreactive, stable amides or, in certain embodiments,as prodrug forms of the compounds according to the present invention.

The term “pharmaceutically acceptable salt” or “salt” is used throughoutthe specification to describe a salt form of one or more of thecompositions herein which are presented to increase the solubility ofthe compound in saline for parenteral delivery or in the gastric juicesof the patient's gastrointestinal tract in order to promote dissolutionand the bioavailability of the compounds. Pharmaceutically acceptablesalts include those derived from pharmaceutically acceptable inorganicor organic bases and acids. Suitable salts include those derived fromalkali metals such as potassium and sodium, alkaline earth metals suchas calcium, magnesium and ammonium salts, among numerous other acidswell known in the pharmaceutical art. Sodium and potassium salts may bepreferred as neutralization salts of carboxylic acids and free acidphosphate containing compositions according to the present invention.The term “salt” shall mean any salt consistent with the use of thecompounds according to the present invention. In the case where thecompounds are used in pharmaceutical indications, including thetreatment of prostate cancer, including metastatic prostate cancer, theterm “salt” shall mean a pharmaceutically acceptable salt, consistentwith the use of the compounds as pharmaceutical agents.

The term “coadministration” shall mean that at least two compounds orcompositions are administered to the patient at the same time, such thateffective amounts or concentrations of each of the two or more compoundsmay be found in the patient at a given point in time. Although compoundsaccording to the present invention may be co-administered to a patientat the same time, the term embraces both administration of two or moreagents at the same time or at different times, provided that effectiveconcentrations of all coadministered compounds or compositions are foundin the subject at a given time. Chimeric antibody-recruiting compoundsaccording to the present invention may be administered with one or moreadditional anti-cancer agents or other agents which are used to treat orameliorate the symptoms of cancer, especially prostate cancer, includingmetastatic prostate cancer. Exemplary anticancer agents which may becoadministered in combination with one or more chimeric compoundsaccording to the present invention include, for example,antimetabolites, inhibitors of topoisomerase I and II, alkylating agentsand microtubule inhibitors (e.g., taxol). Specific anticancer compoundsfor use in the present invention include, for example, Aldesleukin;Alemtuzumab; alitretinoin; allopurinol; altretamine; amifostine;anastrozole; arsenic trioxide; Asparaginase; BCG Live; bexarotenecapsules; bexarotene gel; bleomycin; busulfan intravenous; busulfanoral; calusterone; capecitabine; carboplatin; carmustine; carmustinewith Polifeprosan 20 Implant; celecoxib; chlorambucil; cisplatin;cladribine; cyclophosphamide; cytarabine; cytarabine liposomal;dacarbazine; dactinomycin; actinomycin D; Darbepoetin alfa; daunorubicinliposomal; daunorubicin, daunomycin; Denileukin diftitox, dexrazoxane;docetaxel; doxorubicin; doxorubicin liposomal; Dromostanolonepropionate; Elliott's B Solution; epirubicin; Epoetin alfa estramustine;etoposide phosphate; etoposide (VP-16); exemestane; Filgrastim;floxuridine (intraarterial); fludarabine; fluorouracil (5-FU);fulvestrant; gemtuzumab ozogamicin; goserelin acetate; hydroxyurea;Ibritumomab Tiuxetan; idarubicin; ifosfamide; imatinib mesylate;Interferon alfa-2a; Interferon alfa-2b; irinotecan; letrozole;leucovorin; levamisole; lomustine (CCNU); meclorethamine (nitrogenmustard); megestrol acetate; melphalan (L-PAM); mercaptopurine (6-MP);mesna; methotrexate; methoxsalen; mitomycin C; mitotane; mitoxantrone;nandrolone phenpropionate; Nofetumomab; LOddC; Oprelvekin; oxaliplatin;paclitaxel; pamidronate; pegademase; Pegaspargase; Pegfilgrastim;pentostatin; pipobroman; plicamycin; mithramycin; porfimer sodium;procarbazine; quinacrine; Rasburicase; Rituximab; Sargramostim;streptozocin; talbuvidine (LDT); talc; tamoxifen; temozolomide;teniposide (VM-26); testolactone; thioguanine (6-TG); thiotepa;topotecan; toremifene; Tositumomab; Trastuzumab; tretinoin (ATRA);Uracil Mustard; valrubicin; valtorcitabine (monoval LDC); vinblastine;vinorelbine; zoledronate; and mixtures thereof; among others.

In addition to anticancer agents, a number of other agents may becoadministered with chimeric compounds according to the presentinvention in the treatment of cancer, especially prostate cancer,including metastatic prostate cancer. These include active agents,minerals, vitamins and nutritional supplements which have shown someefficacy in inhibiting prostate cancer tissue or its growth or areotherwise useful in the treatment of prostate cancer. For example, oneor more of dietary selenium, vitamin E, lycopene, soy foods, vitamin D,green tea, omega-3 fatty acids and phytoestrogens, includingbeta-sitosterol, may be utilized in combination with the presentcompounds to treat prostate cancer.

In addition, active agents, other than traditional anticancer agentshave shown some utility in treating prostate cancer. The selectiveestrogen receptor modulator drug toremifene may be used in combinationwith the present compounds to treat cancer, especially prostate cancer,including metastatic prostate cancer. In addition, two medications whichblock the conversion of testosterone to dihydrotestosterone, finasterideand dutasteride, are also useful in the treatment of prostate cancerwhen coadministered with compounds according to the present invention.The phytochemicals indole-3-carbinol and diindolylmethane, may also becoadministered with the present compounds for their effects in treatingprostate cancer. Additional agents which may be combined with compoundsaccording to the present invention include antiandrogens, for example,flutamide, bicalutamide, nilutamide, and cyproterone acetate as well asagents which reduce the production of adrenal androgens (e.g. DHEA),such as ketoconazole and aminoglutethimide. Other active agents whichmay be combined with compounds according to the present inventioninclude, for example, GnRH modulators, including agonists andantagonists. GnRH antagonists suppress the production of LH directly,while GnRH agonists suppress LH through the process of downregulationafter an initial stimulation effect. Abarelix is an example of a GnRHantagonist, while the GnRH agonists include leuprolide, goserelin,triptorelin, and buserelin, among others. These agents may be combinedwith compounds according to the present invention in effective amounts.In addition, abiraterone acetate may also be combined with one or morecompounds according to the present invention in the treatment ofprostate cancer, especially including metastatic prostate cancer.

Other agents which may be combined with one or more compounds accordingto the present invention, include the bisphosphonates such as zoledronicacid, which have been shown to delay skeletal complications such asfractures which occur with patients having metastatic prostate cancer.Alpharadin, another agent, may be combined with compounds according tothe present invention to target bone metastasis. In addition, bone paindue to metastatic prostate cancer may be treated with opioid painrelievers such as morphine and oxycodone, among others, which may becombined with compounds according to the present invention.

The present invention preferably relates to compounds according to thegeneral chemical structure:

Wherein n and n′ are each independently an integer from 1 to 15, often 2to 10, often 2 to 5, more often 2 or 3;NL1 and NL2 are each an integer from 0 to 10, often 1 to 10, often 2 to5, more often 2 or 3, with the proviso that n≧NL1 and n′≧NL2;MCON is an integer from 0 to 10, often 1 to 10, more often 1 to 5, often0, 1 or 2;[IBT] is an immune binding moiety according to the chemical formula setforth in FIG. 14 hereof, preferably, [IBT] is a CP33 group according tothe chemical structure:

[CBT] is a cell binding moiety according to the chemical formula:

Where X₁ and X₂ are each independently CH₂, O, NH or S;X₃ is O, CH₂, NR′, S(O), S(O)₂, —S(O)₂O, —OS(O)₂, or OS(O)₂O;R¹ is H, a C₁-C₃ alkyl group, or a —C(O)(C₁-C₃) group;k is an integer from 0 to 20, 8 to 12, 1 to 15, 1 to 10, 1 to 8, 1 to 6,1, 2, 3, 4, 5 or 6;L₁ and L₂ are each independently a linker according to the chemicalformula:

where R_(a) is H or a C₁-C₃ alkyl, preferably CH₃, most often H;m is an integer from 1 to 12, often 1, 2, 3, 4, 5, or 6;m″ is an integer 1, 2, 3, 4, 5, or 6, often 6;t is 0, 1, 2, 3, 4, 5, or 6; andiL is 0 or 1, often 1, wherein said linker in certain instances, may bepreferably linked to a [CON] group and a [CBT] group at one end and a[MULTICON] group on the other end; or a linker according to thestructure:

Where q is an integer from 0-12, preferably 1, 2, 3, 4, 5 or 6;q′ is 1 to 12, often 1, 2, 3, 4, 5 or 6 andiL is 0 or 1, preferably 1.

The two above linkers may be linked together to provide further linkerswhich are often used in compounds according to the present invention:

Where q is an integer from 0-12, preferably 0, 1, 2, 3, 4, 5 or 6;q′ is 1 to 12, often 1, 2, 3, 4, 5 or 6;iL is 0 or 1; andR_(L) is an amino acid or an oligopeptide (which term includes adipeptide) as otherwise described herein, especially including lysine,dilysine, or glycinelysine.

In certain additional embodiments, the linker group L is an amino acid,a dipeptide or an oligopeptide containing from 1 to 12, preferably 1 to6 amino acid monomers or more. In certain embodiments, the oligopeptideis a dipeptide and the dipeptide is a dilysine or a glycinelysinedipeptide. When lysine is used as an amino acid in an oligopeptidelinker, the sidechain alkylene amine is often used to link other linkergroups or other components in the molecule.

In certain additional embodiments, the linker group L is a group

a group

or a polypropylene glycol or polypropylene-co-polyethylene glycol linkerhaving between 1 and 100 glycol units (1 to 75, 1 to 60, 1 to 55, 1 to50, 1 to 45, 1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to 10, 1 to 8, 1 to6, 1, 2, 3, 4 or 52 and 50, 3 and 45);Where R_(a) is H, C1-C₃ alkyl or alkanol or forms a cyclic ring with R³(proline) and R³ is a side chain derived of an amino acid preferablyselected from the group consisting of alanine (methyl), arginine(propyleneguanidine), asparagine (methylenecarboxyamide), aspartic acid(ethanoic acid), cysteine (thiol, reduced or oxidized di-thiol),glutamine (ethylcarboxyamide), glutamic acid (propanoic acid), glycine(H), histidine (methyleneimidazole), isoleucine (1-methylpropane),leucine (2-methylpropane), lysine (butyleneamine), methionine(ethylmethylthioether), phenylalanine (benzyl), proline (R³ forms acyclic ring with R_(a) and the adjacent nitrogen group to form apyrrolidine group), serine (methanol), threonine (ethanol,1-hydroxyethane), tryptophan (methyleneindole), tyrosine (methylenephenol) or valine (isopropyl);m″ is an integer from 0 to 25, preferably 1 to 10, 1 to 8, 1, 2, 3, 4,5, or 6;m is an integer from 1 to 100, 1 to 75, 1 to 60, 1 to 55, 1 to 50, 1 to45, 1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1, 2,3, 4 or 5; andn is an integer from 1 to 100, 1 to 75, 1 to 60, 1 to 55, 1 to 50, 1 to45, 1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1, 2,3, 4 or 5; orL is a linker according to the chemical formula:

Where Z and Z′ are each independently a bond, —(CH₂)_(i)—O,—(CH₂)_(i)—S, —(CH₂)_(i)—N—R,

wherein said —(CH₂), group, if present in Z or Z′, is bonded to[MULTICON], [ABT], [CBT], or [TLR] or an optional difunctional connectorgroup [CON], if present;Each R is independently H, or a C₁-C₃ alkyl or alkanol group;Each R² is independently H or a C₁-C₃ alkyl group;Each Y is independently a bond, O, S or N—R;Each i is independently 0 to 100, 1 to 100, 1 to 75, 1 to 60, 1 to 55, 1to 50, 1 to 45, 1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to 10, 1 to 8, 1to 6, 1, 2, 3, 4 or 5;

D is

or a bond, or D may be

or a polypropylene glycol or polypropylene-co-polyethylene glycol linkerhaving between 1 and 100 glycol units (1 to 75, 1 to 60, 1 to 55, 1 to50, 1 to 45, 1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to 10, 1 to 8, 1 to6, 1, 2, 3, 4 or 52 and 50, 3 and 45);with the proviso that Z, Z′ and D are not each simultaneously bonds;j is 1 to 100, 1 to 75, 1 to 60, 1 to 55, 1 to 50, 1 to 45, 1 to 40, 2to 35, 3 to 30, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1, 2, 3, 4 or 5;m (within this context) is an integer from 1 to 100, 1 to 75, 1 to 60, 1to 55, 1 to 50, 1 to 45, 1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to 10, 1to 8, 1 to 6, 1, 2, 3, 4 or 5; andn (within this context) is an integer from about 1 to 100, about 1 to75, about 1 to 60, about 1 to 50, about 1 to 45, about 1 to 35, about 1to 25, about 1 to 20, about 1 to 15, 2 to 10, about 4 to 12, about 5 to10, about 4 to 6, about 1 to 8, about 1 to 6, about 1 to 5, about 1 to4, about 1 to 3, etc.).m′ is 1 to 100, 1 to 75, 1 to 60, 1 to 55, 1 to 50, 1 to 45, 1 to 40, 2to 35, 3 to 30, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1, 2, 3, 4 or 5;m″ is an integer between 0 to 25, preferably 1 to 10, 1 to 8, 1, 2, 3,4, 5, or 6;n′ is 1 to 100, 1 to 75, 1 to 60, 1 to 55, 1 to 50, 1 to 45, 1 to 40, 2to 35, 3 to 30, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1, 2, 3, 4 or 5;

X¹ is O, S or N—R;

R is as described above;R_(a) is H, C₁-C₃ alkyl or alkanol or forms a cyclic ring with R³(proline) and R³ is a side chain derived of an amino acid preferablyselected from the group consisting of alanine (methyl), arginine(propyleneguanidine), asparagine (methylenecarboxyamide), aspartic acid(ethanoic acid), cysteine (thiol, reduced or oxidized di-thiol),glutamine (ethylcarboxyamide), glutamic acid (propanoic acid), glycine(H), histidine (methyleneimidazole), isoleucine (1-methylpropane),leucine (2-methylpropane), lysine (butyleneamine), methionine(ethylmethylthioether), phenylalanine (benzyl), proline (R³ forms acyclic ring with R_(a) and the adjacent nitrogen group to form apyrrolidine group), serine (methanol), threonine (ethanol,1-hydroxyethane), tryptophan (methyleneindole), tyrosine (methylenephenol) or valine (isopropyl).[MULTICON] is preferably a multifunctional connector group or moleculeaccording to the chemical structure:

where Y₄ is C—H or N; andEach X″ is independently derived from an electrophilic or nucleophilicgroup, preferably (CH₂)_(n″)O, (CH₂)_(n″)N^(RCON), (CH₂)_(n)″S,(CH₂)_(n″), (CH₂)_(n″)C═O or a [CON] group;the substitutent RCON is H or a C₁-C₃ alkyl, preferably H or CH₃ andn″ is 0, 1, 2 or 3.The optional difunctional connector group or molecule [CON], if present,is a moiety according to the chemical structure:

Where X² is O, S, NR⁴, S(O), S(O)₂, —S(O)₂O, —OS(O)₂, or OS(O)₂O;X³ is NR⁴, O or S; andR⁴ is H, a C₁-C₃ alkyl or alkanol group, or a —C(O)(C₁-C₃) group; ora pharmaceutically acceptable salt, solvate or polymorph thereof.

In preferred aspects of the invention, the immune binding terminus [IBT]is CP33, indicated above.

In preferred aspects of the invention, [CBT] is

Where k is an integer from 0 to 20, 1 to 20, 1 to 8, 2 to 6, 3 to 5, 3to 4,1, 2, 3, 4, 5 or 6.

In certain preferred aspects, the multifunctional connector moiety[MULTICON] is a 1,3,5-triazinyl group according to the structure:

wherein each X″ is independently O, S, C═O, or NR^(CON),and R^(CON) is H or CH₃, preferably H.

In certain preferred aspects, the compound contains a difunctionalconnector moiety [CON} according to the structure:

group which can be covalently bonded at

with a IBT group or a CBT group or alternatively, is preferably bondedto two linker groups to form an extended linker group or directly to a[IBT] or [CBT] group. In certain additional preferred aspects, the [CON]group is

In certain embodiments, [CON] is often a

group;where CL is

m in CL is an integer from 0 to 12, often 0, 1, 2, 3, 4, 5 or 6;and iL is 0 or 1, often 1;In certain embodiments, this [CON] group is often linked through theamine of the triazole to the ring structure of [MULTICON].

In still other aspects the linker group is a oligo or polyethyleneglycolmoiety of the structure:

Where m is from 1 to 100 or as otherwise described herein, preferablyfrom 1 to 12, 2 to 10, 4 to 8, 2 to 6, 8 to 12, 2, 3, 4, 5, 6, 7, 8, 9,10 11 or 12. Noted here is that polypropylene glycol or polyethyleneglycol-co-polypropylene glycol linkers (block copolymers where thepolyethylene glycol portion of the block is from 1 to 12 polyethyleneglycol units in length and said polypropylene glycol portion of theblock is from 1 to 12 polypropylene glycol units in length, the totalnumber of block copolymer units being from 1 to 100, 1 to 50, 1 to 25, 1to 15, 1 to 12, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) may besubstituted for PEG groups in the present compounds. This group may alsobe bonded to two additional linker groups to provided extended linkergroups or directly to one or two functional groups [IBT] and/or [CBT].

Preferred compounds according to the present invention are set forth inattached FIG. 2. Further preferred compounds based upon these compoundspreferably eliminate the biotin moiety of the compounds in FIG. 2 (whichare present for use as reporter in these exemplified compounds) by, forexample, binding the [IBT] group of compound 1 to an amino acid ordipeptide such as glycine or alanine or glycine alanine to provide finalcompounds, or as in compound 2 and 3, by end-capping the lysine group(preferably the carboxylic group with an amine to form the amide asotherwise described herein or the amine with an acyl group, also asdescribed herein).

Pharmaceutical compositions comprising combinations of an effectiveamount of at least one trifunctional chimeric compound SyAM compoundaccording to the present invention, and one or more of the compounds asotherwise described herein, all in effective amounts, in combinationwith a pharmaceutically effective amount of a carrier, additive orexcipient, represents a further aspect of the present invention.

The compositions of the present invention may be formulated in aconventional manner using one or more pharmaceutically acceptablecarriers and may also be administered in controlled-releaseformulations. Pharmaceutically acceptable carriers that may be used inthese pharmaceutical compositions include, but are not limited to, ionexchangers, alumina, aluminum stearate, lecithin, serum proteins, suchas human serum albumin, buffer substances such as phosphates, glycine,sorbic acid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as prolaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

The compositions of the present invention may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional and intracranial injection or infusiontechniques. Preferably, the compositions are administered orally,intraperitoneally or intravenously.

Sterile injectable forms of the compositions of this invention may beaqueous or oleaginous suspension. These suspensions may be formulatedaccording to techniques known in the art using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationmay also be a sterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilmay be employed including synthetic mono- or di-glycerides. Fatty acids,such as oleic acid and its glyceride derivatives are useful in thepreparation of injectables, as are natural pharmaceutically-acceptableoils, such as olive oil or castor oil, especially in theirpolyoxyethylated versions. These oil solutions or suspensions may alsocontain a long-chain alcohol diluent or dispersant, such as Ph. Helv orsimilar alcohol.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, aqueous suspensions or solutions. In thecase of tablets for oral use, carriers which are commonly used includelactose and corn starch. Lubricating agents, such as magnesium stearate,are also typically added. For oral administration in a capsule form,useful diluents include lactose and dried corn starch. When aqueoussuspensions are required for oral use, the active ingredient is combinedwith emulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

Alternatively, the pharmaceutical compositions of this invention may beadministered in the form of suppositories for rectal administration.These can be prepared by mixing the agent with a suitable non-irritatingexcipient which is solid at room temperature but liquid at rectaltemperature and therefore will melt in the rectum to release the drug.Such materials include cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions of this invention may also beadministered topically, especially to treat skin cancers, psoriasis orother diseases which occur in or on the skin. Suitable topicalformulations are readily prepared for each of these areas or organs.Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-acceptable transdermal patches may also be used.

For topical applications, the pharmaceutical compositions may beformulated in a suitable ointment containing the active componentsuspended or dissolved in one or more carriers. Carriers for topicaladministration of the compounds of this invention include, but are notlimited to, mineral oil, liquid petrolatum, white petrolatum, propyleneglycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax andwater.

Alternatively, the pharmaceutical compositions can be formulated in asuitable lotion or cream containing the active components suspended ordissolved in one or more pharmaceutically acceptable carriers. Suitablecarriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the pharmaceutical compositions may be formulated asmicronized suspensions in isotonic, pH adjusted sterile saline, or,preferably, as solutions in isotonic, pH adjusted sterile saline, eitherwith our without a preservative such as benzylalkonium chloride.Alternatively, for ophthalmic uses, the pharmaceutical compositions maybe formulated in an ointment such as petrolatum.

The pharmaceutical compositions of this invention may also beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

The amount of compound in a pharmaceutical composition of the instantinvention that may be combined with the carrier materials to produce asingle dosage form will vary depending upon the host and diseasetreated, the particular mode of administration. Preferably, thecompositions should be formulated to contain between about 0.05milligram to about 750 milligrams or more, more preferably about 1milligram to about 600 milligrams, and even more preferably about 10milligrams to about 500 milligrams of active ingredient, alone or incombination with at least one additional non-antibody attractingcompound which may be used to treat cancer, prostate cancer ormetastatic prostate cancer or a secondary effect or condition thereof.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease or condition beingtreated.

A patient or subject (e.g. a male human) suffering from cancer can betreated by administering to the patient (subject) an effective amount ofa chimeric antibody recruiting compound according to the presentinvention including pharmaceutically acceptable salts, solvates orpolymorphs, thereof optionally in a pharmaceutically acceptable carrieror diluent, either alone, or in combination with other known anticanceror pharmaceutical agents, preferably agents which can assist in treatingprostate cancer, including metastatic prostate cancer or ameliorate thesecondary effects and conditions associated with prostate cancer. Thistreatment can also be administered in conjunction with otherconventional cancer therapies, such as radiation treatment or surgery.

These compounds can be administered by any appropriate route, forexample, orally, parenterally, intravenously, intradermally,subcutaneously, or topically, in liquid, cream, gel, or solid form, orby aerosol form.

The active compound is included in the pharmaceutically acceptablecarrier or diluent in an amount sufficient to deliver to a patient atherapeutically effective amount for the desired indication, withoutcausing serious toxic effects in the patient treated. A preferred doseof the active compound for all of the herein-mentioned conditions is inthe range from about 10 ng/kg to 300 mg/kg, preferably 0.1 to 100 mg/kgper day, more generally 0.5 to about 25 mg per kilogram body weight ofthe recipient/patient per day. A typical topical dosage will range from0.01-3% wt/wt in a suitable carrier.

The compound is conveniently administered in any suitable unit dosageform, including but not limited to one containing less than 1 mg, 1 mgto 3000 mg, preferably 5 to 500 mg of active ingredient per unit dosageform. An oral dosage of about 25-250 mg is often convenient.

The active ingredient is preferably administered to achieve peak plasmaconcentrations of the active compound of about 0.00001-30 mM, preferablyabout 0.1-30 μM. This may be achieved, for example, by the intravenousinjection of a solution or formulation of the active ingredient,optionally in saline, or an aqueous medium or administered as a bolus ofthe active ingredient. Oral administration is also appropriate togenerate effective plasma concentrations of active agent.

The concentration of active compound in the drug composition will dependon absorption, distribution, inactivation, and excretion rates of thedrug as well as other factors known to those of skill in the art. It isto be noted that dosage values will also vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that the concentration ranges set forth herein areexemplary only and are not intended to limit the scope or practice ofthe claimed composition. The active ingredient may be administered atonce, or may be divided into a number of smaller doses to beadministered at varying intervals of time.

Oral compositions will generally include an inert diluent or an ediblecarrier. They may be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound or its prodrug derivative can be incorporated with excipientsand used in the form of tablets, troches, or capsules. Pharmaceuticallycompatible binding agents, and/or adjuvant materials can be included aspart of the composition.

The tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a bindersuch as microcrystalline cellulose, gum tragacanth or gelatin; anexcipient such as starch or lactose, a dispersing agent such as alginicacid, Primogel, or corn starch; a lubricant such as magnesium stearateor Sterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring. When the dosage unitform is a capsule, it can contain, in addition to material of the abovetype, a liquid carrier such as a fatty oil. In addition, dosage unitforms can contain various other materials which modify the physical formof the dosage unit, for example, coatings of sugar, shellac, or entericagents.

The active compound or pharmaceutically acceptable salt thereof can beadministered as a component of an elixir, suspension, syrup, wafer,chewing gum or the like. A syrup may contain, in addition to the activecompounds, sucrose as a sweetening agent and certain preservatives, dyesand colorings and flavors.

The active compound or pharmaceutically acceptable salts thereof canalso be mixed with other active materials that do not impair the desiredaction, or with materials that supplement the desired action, such asother anticancer agents, antibiotics, antifungals, antiinflammatories,or antiviral compounds. In certain preferred aspects of the invention,one or more chimeric antibody-recruiting compound according to thepresent invention is coadministered with another anticancer agent and/oranother bioactive agent, as otherwise described herein.

Solutions or suspensions used for parenteral, intradermal, subcutaneous,or topical application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. The parental preparationcan be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic.

If administered intravenously, preferred carriers are physiologicalsaline or phosphate buffered saline (PBS).

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art.

Liposomal suspensions may also be pharmaceutically acceptable carriers.These may be prepared according to methods known to those skilled in theart, for example, as described in U.S. Pat. No. 4,522,811 (which isincorporated herein by reference in its entirety). For example, liposomeformulations may be prepared by dissolving appropriate lipid(s) (such asstearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline,arachadoyl phosphatidyl choline, and cholesterol) in an inorganicsolvent that is then evaporated, leaving behind a thin film of driedlipid on the surface of the container. An aqueous solution of the activecompound are then introduced into the container. The container is thenswirled by hand to free lipid material from the sides of the containerand to disperse lipid aggregates, thereby forming the liposomalsuspension.

Rationale for Design of Compounds

The first goal in constructing such SyAM-Ps was to design an appropriatebifunctional molecule capable of binding to both the FcγRI receptor andPSMA. We reasoned that linking [CBT] (i.e., PSMA binding motif) to theIBT (i.e., FcγRI binding motif) might allow the bifunctional molecule(SyAM-P1) to mimic an antibody-recruiting molecule, and therefore beable to template ternary complexes between cell-surface PSMA and FcγRI.

PSMA

PSMA is a cell surface protein that is highly overexpressed on prostatecancer cells, in comparison to normal cells of the prostate. Severalsmall-molecule ligands have been developed that bind PSMA selectivelyand with high affinity, including 2-PMPA and the glutamate ureas. The[CBT] (PSMA binding moiety) was chosen accordingly and is used in thepresent invention.

For the immune activation, we chose to target an immune activatingmember of the Fc receptor family, FcγRI, which is expressed on numerouscells of the immune system. In particular, FcγRI is a cell surfaceimmune receptor responsible for initiating pro-inflammatory responsesagainst antibody-opsonized targets. This receptor binds to the Fcportion of IgG, and it is expressed on the surface of numerous immunecells, including monocytes and macrophages. Ligation of this receptorleads to varied pro-inflammatory responses, which include phagocytosisand reactive oxygen species generation. Targets displaying multiplecopies of an FcγRI binding motifs, induce receptor crosslinking andsubsequent signaling, which results in a pro-inflammatory response.¹¹Recently, peptides capable of binding to receptors in the Fc-gammafamily have been reported.^(12,13,14) Specifically, CP33, a peptide thatcan bind to FcγRI, was discovered using phage display.¹² Therefore, theinventors developed the IBT portion of the SyAM preferably using theCP33 peptide to allow selective recruitment and subsequent activation ofeffector cells expressing FcγRI.

Accordingly, the inventors first synthesized the SyAM-P1 molecule, usingFmoc solid phase peptide synthesis to connect the two moieties (FIG. 2).As a consequence of that chemical synthesis the inventors tried todetermine whether linking the two moieties would influence their abilityto bind their respective protein targets, in a selective manner (FIG.3A,B). The inventors thereafter evaluated the ability of thefirst-generation of SyAM-P molecules, identified as SyAM-P1, to bindPSMA on RM1.PGLS cells (FIG. 3A) or FcγRI on IIA1.6 cells (FIG. 3B).These experiments were conducted using SyAM-P1 molecules that werefunctionalized with biotin, and the bound SyAM-P1 was detected using afluorescently labeled streptavidin, via flow-cytometry. SyAM-P1evidenced a capability to bind to its respective targets.

The inventors next evaluated SyAM-P1's ability to induce ternary complexformation, using soluble PSMA and FcγRI expressing cells (IIA1.6 cells)(FIG. 3c ). The binding of PSMA to the cells was probed with aphycoerythrin anti-PSMA antibody, and was analyzed using flow cytometry.The inventors found that the ternary complex formation between FcγRIexpressed on the surface of IIA1.6 cells and soluble recombinant PSMA ismediated through SyAM-P1.

Taken together, this data clearly indicates that SyAM-P1 can interactwith both FcγRI and PSMA simultaneously, thus forming ternary complexesin a cellular milieu. This formation of a ternary complex mimics anantibody's ability to bind its target protein and the Fc receptor. Theseresults serve as critical validation for the general structural designand the computational model, on which it was based. Additionally,connecting the two binding moieties with a hydrophobic linker did notnegate the ability of the individual components from binding to theirrespective partners selectively.

SyAM-P1 was capable of binding to both PSMA and FcγRI selectively, andthus formation of a ternary complex between targets displaying PSMA andprimed effector cells should induce a pro-inflammatory response. Theinventors next tried to determine whether the SyAM-P1 molecule cancrosslink FcγRI and induce an immune response by immune effector cells.To that end, to evaluate the pro-inflammatory responses mediated by SyAMmolecules, the inventors analyzed the superoxide burst and phagocyticresponses of immune effector cells. (FIG. 3 D,E). The inventors used animmortalized monocytic effector cell line (U937 cells) to evaluate theSyAM-P1-mediated immune effector responses. 937 cells were primed withIFN-γ, which induces an upregulation of FcγRI and primes the cells forthe pro-inflammatory responses (superoxide burst and phagocytosis).Superoxide burst is characterized by the release of reactive oxygenspecies (ROS) that can occur during pro-inflammatory responses.

The magnitude of this response can be measured using the lucigeninassay. Phagocytosis is the active engulfment of an opsonized target.The phagocytosis assay used involved incubating PSMA labeled F11fluorescent beads with F14 stained IFN-γ primed U937 cells, in thepresence of SyAM-P1. Phagocytosis was observed by flow cytometry andmicroscopy. The results indicated that SyAM-P1 was able to inducesuperoxide burst (FIG. 3D) and phagocytosis of PSMA coated beads (FIG.3E) in a dose-dependent manner. In particular, SyAM-P1 was able toinduce superoxide burst at concentrations of 100 nM and in a targetdependent manner. In the phagocytosis assay, the compound had an EC₅₀ of26 nM. These immune effector responses identified above were abrogated,when the assays were conducted in the presence of either a PSMAinhibitor (2-PMPA) or FcγRI inhibitor (IgG). (data not shown)

The results evidenced that the ternary complex binding resultstranslated into active immunological responses. These proof of principleexperiments for SyAM-P1 supported the hypothesis that a fully syntheticmolecule might induce an FcγRI-dependent immune response against targetsdisplaying PSMA. Both the targeting and effector functions of anantibody could be effectively mimicked by a fully synthetic bifunctionalmolecule.

Since SyAM-P1 was able to induce effector-cell mediated responsesagainst target protein coated beads, the inventors then evaluated if themolecule can elicit the effects against PSMA expressing cells. To thatend, the inventors evaluated the SyAM-P1 induced immune responses byprimed U937 effector cells against PSMA expressing RM1.PGLS cells. Theytested the U937-mediated immune effects by evaluating both superoxideburst and phagocytosis of effector cells, analogous to the proteincoated bead experiments. In this experiment, IFN-γ primed U937 cellswere incubated with PSMA expressing RM1.PGLS cells, and treated withvarious concentrations of SyAM-P1. The resulting superoxide burstresponse was measured using the lucigenin assay (data not shown).Additionally, primed U937 cells (stained with DiD, an FL4 membrane dye)were incubated with target RM1.PGLS cells (stained with DiI, an FL1membrane dye), in the presence of various concentrations of SyAM-P 1.The resulting phagocytosis—indicated by FL1 and FL4 double positivecells—was measured using two-color flow-cytometry. As a positivecontrol, ARM-P8 combined with anti-DNP antibodies was utilized. WhileARM-P8 was capable of inducing significant responses, SyAM-P1 was unableto induce any immune responses against the target cells (data notshown). The positive response seen with the Arm-P8 control, couldpotentially be attributed to the ability of antibodies to ligate bothFcγRI and FcγRIIA on the immune effector cells, increasing immunesignaling and phagocytic responses.

Given that SyAM-P1 molecule was capable of inducing effectorcell-mediated phagocytosis against beads labeled with PSMA, yet unableto elicit similar responses against PSMA expressing cells, wehypothesized that this may be a result of differential levels of PSMAmolecules per μm² of surface area of the two different targets. (FIG.5). These results prompted the inventors to compare the PSMA labeling onthe surface of beads, with that of RM1.PGLS cells.

The level of opsonization of the target cell is directly proportional tothe effector cell-mediated immune response observed, and importantly, athreshold opsonization must be reached prior to observing any response.The ability of SyAM-P1 to mediate phagocytosis was assessed utilizingbeads loaded with various amounts of PSMA. The level of phagocytosis ofbeads by primed U937 cells directly correlated to the level of PSMAdisplayed on the surface. (FIG. 5). The level of expression of PSMA onthe surface of RM1.PGLS cells was measured with a phycoerythrinanti-PSMA antibody and calculated with phycoerythrin fluorescentcalibration beads. RM1.PGLS cells display a fewer PSMA proteins per μmthan the lowest level of bead tested. (FIG. 5). RM1.PGLS cells displayedapproximately 918 PSMA proteins/um², while the beads used in the assaydisplayed approximately 5577 PSMA proteins/um². Given the directcorrelation between effector responses and the level of opsonization,the level of PSMA expression on the surface of the target cells wasinsufficient to induce a SyAM-P1-mediated response. Given that the levelof PSMA on the surface directly correlates to the efficacy of theSyAM-P1 molecule, the inventors then hypothesized that increasing theaffinity of the TBT region of the molecule, through bivalent display,might increase the amount of SyAM-P1 molecules binding to the surfacewith PSMA, at equilibrium. The inventors reasoned that: by increasingthe local concentration of the PSMA binding moiety, the apparent Kd willbe increased, which would enhance the range of effective concentrationsand increase the level of molecule displayed at equilibrium.

Pursuant to that hypothesis, the SyAM-P molecule was redesigned toincorporate a bivalent display of PSMA targeting motifs in order toenhance its efficacy (SyAM-P2) (FIG. 2, compound 2). First, theyverified the binding and compared the affinity of SyAM-P2 to PSMA coatedbeads. Thereafter, SyAM-P2-mediated immune responses of primed U937cells against PSMA coated beads was evaluated. (FIG. 4A). Finally, theinventors tested the effects of SyAM-P2 on its ability to induce asuperoxide burst response or phagocytic response in primed U937 cells,against RM1.PGLS cells. (Data not shown). The binding of SyAM-P1 to PSMAcoated beads (EC₅₀=40 nM) were less effective than that of SyAM-P2(EC₅₀=26 nM).

SyAM-P2 was able to induce a much higher level of phagocytosis (SI FIG.2A) of PSMA coated beads, with a wider efficacy range, in comparison toSyAM-P1. Encouraged by these results, the inventors proceeded toevaluate the ability of SyAM-P2 to mediate immunological effects againstRM1.PGLS cells. Unfortunately, no response was seen with eithersuperoxide burst or phagocytosis (data not shown). However, increasingthe display of binding motifs, directly increased the level ofsuperoxide burst response and phagocytosis against PSMA coated beads.This verified the hypothesis that increasing the valency on the PSMAbinding side will increase the apparent Kd (SI FIG. 4). The level ofligation of the FcγRI by SyAM-P2, in the presence of cells, is stillinadequate to induce a favorable, clinically relevant immune responseagainst PSMA expressing cells.

The inventors also explored linker length and composition in this secondgeneration molecule, which showed the most robust results when utilizingthe aminocaproic acid linker displayed in SyAM-P2 (SI FIG. 3). Thesecond generation of SyAM molecules increased the efficacy of thePSMA-bead experiments, but still proved unable to induce a favorableresponse against RM1.PGLS cells. The inventors hypothesized that this isdue to insufficient crosslinking of the Fc receptor. Considering this,the inventors optimized SyAM-P2 to enhance its efficacy through both abivalent display of PSMA targeting motif and the FcγRI targeting motif;this molecule was named SyAM-P3 (FIGS. 1, 3).

The inventors then tested SyAM-P3-induced immune effects (i.e.,U937-mediated superoxide burst and phagocytosis) against PSMA coatedbeads (FIGS. 4A and B). SyAM-P3 was able to induce a much greater immuneresponse against PSMA-beads, in both the generation of superoxide burst(FIG. 4A) and phagocytosis (FIG. 4b ) as compared to SyAM-P2; theresponse was greater in both potency of phagocytosis and the efficaciousrange. In control experiments superoxide burst was shown to be dependenton the presence of both molecule and PSMA labeling of the bead (SI FIG.6). Phagocytosis of beads could compete with both 2-PMPA and human IgG(SI FIG. 5). Finally, the inventors also evaluated the efficacy ofSyAM-P3 on facilitating U937-mediated phagocytosis of PSMA expressingRM1.PGLS cells (FIG. 4C). In this case, SyAM-P3 was able to effectivelyinduce U937-mediated phagocytosis, in a dose dependent manner, againstRM1.PGLS cells (FIG. 4C). The inventors were able to visually verify thephagocytosis of RM1.PGLS cells, using a flow cytometer (Amnis) withimage acquisition capabilities (FIG. 4D). Using this method, theinventors visualized both early stages of phagocytosis, where thephagocytic cup is being formed, and late stages of complete phagocytosedRM1.PGLS. This phagocytosis could compete with both 2-PMPA and human IgG(SI FIG. 7). No response was seen with SyAM-P3 and RM1.PGLS cells in thesuperoxide burst assay.

Based on the ternary complex model, [ref] (SI FIG. 4B) increasing thenumber of FcγRI ligating motifs, enhanced both the potency and efficacyof SyAM-P molecules to elicit targeted immune responses against PSMAcoated beads. SyAM-P3 is effective at stimulating immune effectorcell-mediated phagocytosis of PSMA expressing cells. The response withSyAM-P3 peaked at 10 nM concentration of the molecule, which is a shiftfrom the maximal response peak observed with the use of ARM-P8 andanti-DNP antibodies (SI FIG. 8). This shift in the ternary complexequilibrium maximum shows our molecule can induce an immunologicalresponse at a concentration an order of magnitude better than the ARM-P8molecule.

Discussion

The compounds disclosed in the present application report are unique, inthat they are the first reported fully synthetic molecules capable ofdirectly mimicking a monoclonal antibody's ability to induce an immuneresponse through FcγRI in a highly specific manner. Given that the SyAMsof the present invention are synthetic molecules capable of eliciting animmune response by directly binding to immune cells, they can mediatetheir functions independent of endogenous antibodies, unlike the ARMmolecules.^(8b, c) The SyAM molecules of the present invention functionthrough a single activating receptor, FcγRI, limiting the crossreactivity with other arms of the immune system. The specificity of themolecule for FcγRI prevents cross ligation of the inhibitory FcγRIIb,which has been implicated in lowering the efficacy of mAbs in-vivo. Byselectively targeting the Fc family of receptors, there exists thepotential for cross presentation of antigenic sequences and to elicitfurther adaptive immune responses. The SyAMs specifically address theunfavorable aspects with mAbs such as, the lack of chemical homogeneity,the limited scope of pathogen target, activation of complementdeposition impairing Fc responses,¹⁵ and the difficulty in predictingthe mechanism of action in vitro.

In addition to being easier to synthesize and purify, the scaffold andconvergent synthesis of the present invention lends itself to rapidmodification. This allows us to append other TBTs rapidly, thusbroadening the scope of the utility of the approach. More broadly, thegeneral strategy for using small molecules to redirect the cytotoxicfunctions of immune cells has the potential for application in widerange of pathophysiologically unrelated human diseases, such as viraland bacterial infection.

Chemical Synthesis

The chemical synthesis of compounds according to the present inventionproceeds stepwise by preparing building blocks of various components andthen condensing these building blocks onto each other to fashioncompounds according to the present invention. While the specificsyntheses of compounds identified in the present patent applicationproceeds in a step-wise fashion with individual components, varioussubstitutions which are used may be readily substituted for othercomponents using analogous chemistries to synthesize all of thecompounds of the present invention. The following synthetic schemes areexemplary of the chemistry which is used to prepare compounds accordingto the present invention. This chemistry may be used directly or in ananalogous fashion to prepare all of the compounds of the presentinvention.

SyAM-P1 (with Biotin)

This compound is synthesized pursuant to the chemical synthesis schemewhich is presented in FIG. 15 to provide SyAM-P1 of FIG. 16. Pursuant tothe scheme presented in FIG. 15, the triester urea CBT group containingan azide group (presented in the reaction scheme for SyAM-P3hereinbelow) is reacted with the acetylenic hexynoic acid in sodiumascorbate, copper sulfate, water, tert-butanol at elevate temperature ina microwave to form the triazole [CON] group which is covalentlyattached to [CBT]. The free carboxylic acid group of that intermediateis then converted to an acyl chloride derivative, which is reacted withthe lysine urethane derivative in DIPEA to form the CBT-lysineintermediate containing the urethane moiety. Solid phase peptidesynthesis places a glycine group on the carboxyl acid of the lysine andthe amine group is deprotected and substituted with a peptide linkercomprising amino hexanoic acid units (FIG. 2 shows 5 amino hexanoic acidunits). This intermediate is further reacted with a CP33-linked lysinegroup (if biotin attachment is desired) or a CP33-amino acid (alanine orglycine) to link the CP33 group to the CBT moiety molecule (withoutbiotin attachment). Alternatively, the CP33 group can be linked toanother amino acid, an oligopeptide, including a dipeptide (such asglycine alanine, glycine glycine or alanine alanine, among numerousother combinations) to provide a SyAM-P1 molecule.

SyAM-P2

The synthesis of SyAM-P2 proceeds pursuant to the reaction scheme whichis presented in FIG. 17. Pursuant to that synthesis, the triesterblocked glutamate-lysine dipeptide containing the azido group on thesidechain of lysine is reacted with amino end-capped polyethylene glycolcontaining an acetylenic group to form the intermediate CBT complex witha free amine (s-2 of FIG. 17). The free amine is reacted with the[MULTICON] moiety (s-3 of FIG. 17) to condense two CBT groups onto the[MULTICON] moiety S-3 as indicated in step b. The resulting intermediatecomprises two CBT groups, a linker connecting the [CON] groups of X-2 tothe [MULTICON] group (s-3) to provide intermediate S-4 (which can varyas to the substitution on the linker attached to triazole [CON] group)which can be condensed with intermediate 2-7 which contains a CP33 grouplinked to a dipeptide or oligopeptide. The dipeptide or oligopeptide maybe end-capped to provide a non-reactive group or further reacted toprovide a biotin moiety for diagnostic/experimental applications. S-7 inFIG. 17 links CP33 through an alanine-lysine-lysine tripeptide whereinthe side chain of the distal lysine binds to the biotin moiety, butmodifications of S-7 may be readily made. Alternatively, SyAM-P2 whichavoids biotin can simply have the first lysine end-capped with anon-reactive group (e.g. amide) rather than extending the compound toanother lysine to which is attached the biotin moiety. In the chemicalsynthesis of FIG. 17, the [IBT] group (CP33 containing intermediate S-7)is condensed onto the activated ester of intermediate s-4 to link theCP33 containing component to the [MULTICON] group to which are bondedthe two CBT linker groups (s-2). This results in the final productSyAM-P2, which can contain a biotin group or other reporter componentfor diagnostics/analysis or can avoid the presence of a reportercomponent for use as a therapeutic agent in the treatment of prostatecancer as otherwise described herein.

SyAM-P3 (without Biotin)

This is the preferred compound pursuant to the present invention. Thiscompound has two CBT groups and two IBT groups which are linked througha central {MULTICON] trifunctional 1,3,5-phenyl group. The synthesis ofthis compound begins with the preparation of the preferred CBT group(under the heading urea formation) pursuant to the chemical syntheticscheme which is presented in FIG. 17 hereof. The diester protectedglutamate analog is first reacted with triphosgene followed by adi-protected lysine compound (the α-amino group remains unprotected) toform the tetra-protected intermediate which is hydrogenated (pd/C) toremove the Cbz protecting group at the sidechain amine position oflysine, which is converted to an azide moiety using TfN₃, copper sulfateand potassium carbonate to form the triprotected urea containing anazide moiety. This compound is the CBT synthon used in much of thesynthesis of the present compounds because the azide can be condensedwith an acetylenic group to readily form a triazole [CON] group which isdirected bonded to the preferred [CBT] moiety in compounds according tothe present invention.

In the same FIG. 17, the unnatural amino linker is prepared from thesynthetic diprotected amino acid compound by removing the Boc groupwhich protects the amine functionality in strong acid in dioxane,followed by condensing an amine protected amino acid onto the free aminoof the amine-deprotected amino acid in1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (EDC),1-hydroxybenzotriazole (HOBt) and N,N-diisopropylethyl amine (DIPEA) toprovide the benzyl protected tripeptide. The terminal amine group of theresulting tripeptide is then protected (Fmoc) and the benzyl group isdeprotected using hydrogenation conditions to afford the N-terminalprotected linker tripeptide. This is the first linker.

The second linker is synthesized by condensing an acetylenic amine grouponto the free carboxylic acid group of the di-N protected (Fmoc) lysinecompound in the presence of EDC, HOBt and DIPEA. The resultingacetylenic protected lysine compound is then deprotected and reactedwith the amine protected first linker obtained above to form thedilinker substituted acetylenic lysine intermediate which is deprotected(the two Fmoc groups are removed in diethylamine) and subsequentlycondensed with the azido dicarboxylic acid benzene analog. The benzeneanalog (which becomes the [MULTICON] group is then reacted with thebenzyl-protected tripeptide linker in EDC, HOBt, DIPEA to form thephenyl azide containing two tripeptide linking groups. The azido-cappedurea moiety [CBT] prepared above is condensed with the acetylenic aminecompound to form the triazole [CON] group on the azide position having amethyleneamine group. This intermediate is reacted with the amineprotected tripeptide linker to condense the tripeptide linker onto thefree amine group of the triazole moiety. The protecting group (Fmoc) onthe amine terminus of the tripeptide linker is removed withdiethylamine. This intermediate is then condensed onto the dilinkerazido phenyl [MULTICON] intermediate to provide two CBT linker groupswhich have been condensed onto the azido phenyl [MULTICON] intermediate.The azido group on the phenyl intermediate is condensed onto theacetylenic moiety of the second linker, prepared above to form atriazole group as the third group on the phenyl [MULTICON] moiety. Thisintermediate contains two CBT-linker groups on the phenyl [MULTICON]moiety and a triazole [CON] moiety on the [MULTICON] moiety. Thetriazole [CON] moiety also contains the two free amine-triethyleneglycol linkers linked through lysine and end-capped with amine groupswhich can condense with CP33 to produce the final SyAM-P3 compound.

Accordingly, CP33 is prepared for condensation onto the two free aminegroups by reacting a di-blocked lysine intermediate used in thepreparation of the CBT urea group with the free carboxylic acid end ofCP33 to form a lysine reacted CP33 intermediate which undergoesamination to form the free amide with the unreacted carboxylic acidgroup of lysine group. The free amine of lysine on the CP33-lysineintermediate is reacted with the disuccinimido diketo linker precursorto form the CP33-lysine linked intermediate containing a succinimidegroup (as a leaving group). Two of these CP33-lysine linkedintermediates are condensed onto the previously prepared intermediatecontaining the two CBT groups which are linked through the [MULTICON]phenyl group and contain two free amine groups which readily condenseonto the CP33-lysine linked intermediate containing thesuccinimido-activated ester group forming the final compound SyAM-P3 asset forth in FIG. 23.

-   -   Alternative approaches to the synthesis of compounds according        to the present invention are described in FIG. 24. FIG. 24 shows        alternative syntheses of SyAM-P2 which can be applied to other        compounds according to the present invention, utilizing        alternative linkers as set forth in the figure. All of the        reactions are straight forward and result in numerous compounds        which can be seen to vary with respect to the linkers used.

Examples General Information Synthesis:

All starting materials and reagents were purchased from commerciallyavailable sources and used without further purification. ¹H NMR shiftsare measured using the solvent residual peak as the internal standard(CDCl₃ d 7.26, MeOD d 3.31), and reported as follows: chemical shift,multiplicity (s=singlet, bs=broad singlet, d=doublet, t=triplet,dd=doublet of doublet, dt=doublet of triplet, q=quartet, m=multiplet),coupling constant (Hz), integration. ¹³C NMR shifts are measured usingthe solvent residual peak as the internal standard (CDCl₃ d 77.20, MeODd 49.00, or DMSO d 39.52), and reported as chemical shifts. Infrared(IR) spectral bands are characterized as broad (br), strong (s), medium(m), and weak (w).

ABBREVIATIONS

AcOH=acetic acid

Acn=Acetonitrile

Ahx=Aminocaproic acidAMC=7-amino-4-methylcoumarinBoc=tert-butoxycarbonylBSA=bovine serum albuminCbz=benzyloxycarbonylDCM=dichloromethaneDIPEA=diisopropylethyl amine

DMF=N,N-dimethylformamide

DMSO=dimethylsulfoxideDPBS=Dulbecco's phosphate-buffered salineEDC=1-ethyl-3-(3-dimethylaminopropyl) carbodiimideEDTA=ethylenediaminetetraacetic acid, disodium saltEGTA=ethylene glycol-bis(2-aminoethyl)-N,N,N′,N′-tetraacetic acidEtOAc=ethyl acetateFmoc=9-fluorenylmethyloxycarbonylHI-FBS=heat inactivated fetal bovine serumHOBt=hydroxybenzotriazoleiPrOH=isopropyl alcoholMeCN=acetonitrileMeOH=methanolMTT=methyl-trityl

NHS=N-hydroxysuccinimide NMP=N-methylpyrollidinone

Pbf=2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyPd/C=10% palladium on carbonQuant.=quantitative conversionTBS=Tris-buffered salineTbu=tertbutylTEA=triethylamine.TFA=trifluoroacetic acidTFAA=trifluoroacetic anhydrideTHF=tetrahydrofuraneTiPS=triisopropylsilaneTrt=trityl

Solid Phase Peptide Synthesis General Procedure:

Solid-Phase Peptide Synthesis (SPPS) was performed in a CEM DiscoverLiberty Microwave Peptide Synthesizer. The amount of resin, amino acids,and reagents used in the synthesis were calculated using manufacturesuggested protocols based on 0.1 mmol scale synthesis. Upon completionof SPPS, the resin was collected through vacuum filtration and rinsedseveral times with CH2Cl2. After drying in open air, the resin was thenadded to a flask containing either cleavage cocktail mixture (92:4:4mixture of TFA:TIPS:H₂O) and gently stirred for the time specifiedbelow. Subsequently, the resin was filtered through a cotton-pluggedpipet, and the filtrate was directly collected into a 50 mL conical tubecontaining 35 mL of cold (−78° C.) Et2O, at which point a whiteprecipitate immediately formed. The suspension was re-cooled to −78° C.and then centrifuged at 4400 rpm for 5 minutes. After decanting thesupernatant, the residual pellet was taken up in 50% MeCN/H₂O andpurified using reverse-phase HPLC in 6-7 portions. The HPLC fractionswere combined and lyophilized to give the corresponding peptides aswhite fluffy material.

Synthesis

5-(1-((R)-6-(tert-butoxy)-5-(3-((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)ureido)-6-oxohexyl)-1H-1,2,3-triazol-4-yl)pentanoicacid

A mixture of S.1 (98 mg, 0.191 mmol, 1.0 equiv.) and heptynoic acid (120mg, 0.954 mmol, 5 eq.) was dissolved in a mixture of H₂O (1.25 mL) andt-BuOH (1.25 mL) in a 5 mL □wave reaction tube. To this mixture wasadded 0.1 M sodium ascorbate (0.059 mmol, 0.2 equiv.) and 0.1 M copper(II) sulfate (0.012 mmol, 0.04 equiv.). The tube was capped, andsubjected to □wave irradiation for 10 minutes at 110° C. The reactionwas then concentrated under reduced pressure, and chromatographed (1×15cm silica gel, 20% MeOH in CHCl₃, then 20% MeOH in CHCl₃+1% TFA) toyield s-x as a colorless oil calc'd for C₃₁H₅₃N₅O₉ (M+H) 640.7806 found.

SvAM-P1

Standard solid phase peptide synthesis 0.1 mmol scale ofFmoc-VNS(Tbu)C(Tr)LLLPN(Trt)LLGC(Tr)GD(Tbu)D(Tbu)K(biotin)-Ahx₅-Lys(MTT)-G-Resin.While on resin Mtt group deprotected using 1% TFA in DCM 5 mL while onrotator for 5 minutes, repeat three times, a yellow supernatant washedaway. Resin neutralized washing with DMF 0.1 mM DIPEA. Sx (160 mg, 0.25mmol, 2.5 eq) dissolved in 3 mL of DMF and added to resin with HBTU (95mg, 0.25 mmol, 2.5 eq) along with 86 uL of DIPEA (64 mg, 5 mmol, 5 eq).Mixture subjected to μwave irradiation for 10 minutes at 75c. Resinwashed 3× with DMF and Fmoc group deprotected with 20% piperidine in DMFfor 20 minutes RT. Global deprotection and cleavage from solid supportperformed with 92:4:4 mixture of TFA:H₂O:TIPS for 90 minutes stirring atroom temperature. Subsequently, the resin was filtered through acotton-plugged pipet, and the filtrate was directly collected into a 50mL conical tube containing 35 mL of cold (−78° C.) Et2O, at which pointa white precipitate immediately formed. The suspension was re-cooled to−78° C. and then centrifuged at 4400 rpm for 5 minutes. After decantingthe supernatant, the residual pellet was taken up in 25 mL of 20%ACN/H₂O and 1 mL of DMSO and 40 mg of potassium carbonate added andstirred in air for 48 hours to oxidize to disulfide. After oxidationpeptide was purified using reverse-phase HPLC.

HRMS (ES+) calc'd for C₁₄₂H₂₃₈N₃₆O₄₁S₃ (M+3H) m/z 1601.91 found (M+3H)1601.53.

(S)-di-tert-butyl2-(3-((S)-6-(4-(25-amino-2,5,8,11,14,17,20,23-octaoxapentacosyl)-1H-1,2,3-triazol-1-yl)-1-(tert-butoxy)-1-oxohexan-2-yl)ureido)pentanedioate(s-2)

A mixture of s-1 (152 mg, 0.295 mmol, 1.0 equiv.) and3,6,9,12,15,18,21,24-octaoxaheptacos-26-yn-1-amine (120 mg, 0.295 mmol,1.0 equiv.) was dissolved in a mixture of H₂O (1.1 mL) and t-butanol(1.1 mL) in a 5 mL μwave reaction tube. To this mixture was added 0.1 Msodium ascorbate (0.6 mL, 0.2 equiv.) and 0.1 M copper(II) sulfate (0.12mL, 0.04 equiv.). The tube was capped, and subject to μwave radiationfor 2.5 minutes at 110° C. The reaction was then concentrated underreduced pressure, and chromatographed (1×15 cm silica gel, 10% MeOH inCH₂Cl₂, then 10% MeOH in CH₂Cl₂+2.5% Et₃N) to yield s-2 (254 mg, 80%) asa brown oil. IR (thin film) 2869 (m), 1729 (s), 1680 (w), 1534 (m), 1456(w), 1367 (m), 1252 (w), 1152 (s), 1113 (s) cm-1. 1HNMR (400 MHz, CDCl3)d 7.71 (s, 1H), 5.32 (d, J=8 Hz, 1H), 5.25 (d, J=8 Hz, 1H), 4.72-4.64(dd, J=8 Hz, XX Hz, 2H), 4.38-4.28 (m, 4H), 3.68-3.64 (m, 30H),2.98-2.93 (m, 2H), 2.35-2.28 (m, 2H), 2.08-2.05 (m, 1H), 1.97-1.77 (m,4H), 1.65-1.59 (m, 1H), 1.46 (s, 9H), 1.43 (s, 18H), 1.49-1.27 (m, 2H).13CNMR (100 MHz, CDCl3) d 172.5, 172.1, 156.9, 145.0, 123.0, 81.9, 81.9,90.5, 70.6, 70.5, 70.5, 70.5, 70.4, 70.4, 70.4, 70.2, 69.6, 64.6, 53.1,53.0, 49.9, 32.3, 31.7, 29.6, 28.3, 28.1, 28.1, 28.0, 21.9. HRMS (ES+)calc'd for C₄₃H₈₀N₆O₁₅ (M+H) m/z 921.5754. Found 921.5754.

5-(4-(5-methoxy-5-oxopentyl)-1H-1,2,3-triazol-1-yl)isophthalic acid(s-3)

A mixture of azidoisophthalic acid (100 mg, 0.483 mmol, 1.0 equiv.) andmethyl hept-6-ynoate (100 mg, 0.714 mmol, 1.45 equiv.) was dissolved ina mixture of H₂O (1.7 mL) and t-BuOH (1.7 mL) in a 5 mL μwave reactiontube. To this mixture was added 0.1 M sodium ascorbate (0.059 mmol, 0.2equiv.) and 0.1 M copper (II) sulfate (0.012 mmol, 0.04 equiv.). Thetube was capped, and subjected to μwave irradiation for 2.5 minutes at110° C. The reaction was then concentrated under reduced pressure, andchromatographed (1×15 cm silica gel, 20% MeOH in CHCl₃, then 20% MeOH inCHCl₃+1% TFA) to yield s-3 as a beige colored solid. IR (thin film) 3151(w), 2951 (w), 1721 (s), 1604 (w), 1463 (w), 1291 (m), 1248 (m), 1071(m) cm-1. 1HNMR (400 MHz, MeOD) d 8.70 (s, 1H), 8.66 (s, 2H), 8.51 (s,1H), 3.66 (s, 3H), 2.83 (t, J=7.4 Hz, 2H), 2.41 (t, J=6.8 Hz, 2H),1.81-1.70 (m, 4H). 13CNMR (100 MHz, DMSO-d₆) d 173.3, 165.9, 148.3,137.2, 133.5, 129.1, 123.7, 120.6, 118.0, 51.2, 33.0, 27.9, 24.7, 24.0.HRMS (ES+) calc'd for C₁₆H₁₇N₃O₆ (M+H) m/z 348.1190. Found 348.1184.

s-4

To a solution of s-3 (20 mg, 0.06 mmol, 1 equiv.) in 1 mL of dry CH₂Cl₂in a flame dried round bottom flask was added EDC (29 mg, 0.15 mmol, 2.5equiv.) and HOBt (23 mg, 0.15 mmol, 2.5 equiv.), followed by a solutionof s-2 (122 mg, 0.133 mmol, 2.2 equiv.) in 1 mL of dry CH₂Cl₂ andpyridine (15 uL, 0.181 mmol, 3 equiv.). The reaction was allowed to stirat room temperature for 13 hours, after which it was concentrated underreduced pressure at 37° C. and chromatographed (silica gel, 2×15 cm, 5%MeOH in CHCl₃) to give s-4 as a pale brown oil (90 mg, 73%). IR (thinfilm) 2867 (w), 2405 (br), 1728 (s), 1661 (m), 1456 (s), 1367 (m), 1250(w), 1149 (s), 1098 (s), 846 (w) cm-1. 1HNMR (500 MHz, MeOD) d 8.50 (s,1H), 8.50 (s, 1H), 8.42 (t, J=1.5 Hz, 1H), 7.96 (s, 2H), 6.36-6.33 (dd,J=4, 4.5 Hz, 3H), 4.60 (s, 4H), 4.40 (t, J=7.5 Hz, 4H), 4.22-4.11 (m,4H), 3.70 (t, J=5.5 Hz, 4H), 3.67-3.54 (m, 66H), 2.83 (t, J=7 Hz, 2H),2.40 (t, J=7 Hz, 2H), 2.37-2.27 (m, 4H), 2.07-2.00 (m, 2H), 1.96-1.89(m, 4H), 1.84-1.70 (m, 8H), 1.69-1.61 (m, 2H), 1.47 (s, 19H), 1.44 (s,21H), 1.41 (s, 17H), 1.40-1.34 (m, 4H). 13CNMR (100 MHz, MeOD) d 175.6,173.7, 173.6, 173.4, 167.9, 159.9, 150.1, 146.0, 138.7, 138.0, 127.4,125.0, 122.8, 121.8, 82.8, 82.6, 81.7, 71.6, 71.6, 71.5, 71.3, 70.8,70.4, 65.0, 54.7, 54.1, 52.1, 51.1, 41.3, 34.4, 32.9, 32.5, 30.8, 29.8,29.0, 28.4, 28.3, 26.0, 23.5. HRMS (ES+) calc'd for C₁₀₂H₁₇₃N₁₅O₃₄ (M+H)2153.2369. Found (M+2H) m/z 1077.1271.

s-5

s-4 (90 mg, 0.042 mmol, 1 equiv.) was dissolved in MeOH (1 mL). To thissolution was added 1 M LiOH in H₂O (4 equiv.). The reaction was allowedto stir at room temperature for 2 hours, after which another 4 equiv. of1 M LiOH in H₂O was added. The reaction was allowed to stir at roomtemperature fore another 2 hours, after which it was neutralized with 6equiv. of 1 M HCl in H₂O. The reaction was concentrated andchromatographed (1×15 cm silica gel, 20% MeOH in CHCl₃). The fractionscontaining product were concentrated and taken up with 80% MeCN in H₂Oand purified using HPLC (C18 reverse phase, 5 mL/min, 50%-75% MeCN inH₂O over 40 minutes). The product was isolated and lyophilized to give as-5 as a white powder (10 mg, 12%). IR (thin film) 3341 (br), 3108 (w),2931 (m), 1729 (s), 1667 (s), 1556 (w), 1457 (w), 1151 (s) cm-1. 1HNMR(500 MHz, MeOD) d 8.73 (t, J=5.5 Hz, 1H), 8.51 (s, 3H), 8.42 (s, 1H),7.97 (s, 2H), 4.64 (s, 1H), 4.60 (s, 4H), 4.41 (t, J=7.2 Hz, 4H), 4.18(dd, J=5, 9 Hz, 2H), 4.13 (dd, J=5, 8 Hz, 2H), 4.06 (s, 1H), 4.01 (s,1H), 3.99-3.96 (m, 1H), 3.74-3.55 (m, 80H), 3.48-3.44 (m, 1H), 3.17-3.13(m, 1H), 2.84 (t, J=7.2 Hz, 2H), 2.66 (s, 1H), 2.39-2.29 (m, 6H),2.07-2.00 (m, 2H), 1.96-1.89 (m, 4H), 1.84-1.76 (m, 6H), 1.75-1.70 (m,2H), 1.69-1.61 (m, 2H), 1.60-1.52 (m, 2H), 1.48-1.41 (m, 4H), 1.47 (s,18H), 1.44 (s, 18H), 1.43 (s, 18H), 1.40-1.30 (m, 6H). 13CNMR (125 MHz,MeOD) d 177.2, 173.7, 173.7, 173.4, 168.0, 159.9, 138.8, 138.0, 127.4,125.0, 122.7, 121.8, 82.8, 81.8, 71.5, 71.4, 71.4, 71.4, 71.3, 70.7,70.5, 65.0, 54.7, 54.2, 41.3, 34.6, 32.9, 32.5, 30.8, 29.8, 29.0, 28.4,28.3, 26.1, 25.5, 23.5. HRMS (ES+) calc'd for C₁₀₁H₁₇₁N₁₅O₃₄ (M+H) m/z2139.2213. Found (M+2H) m/z 1070.1426.

cp33-Bis(Peg8-Urea)

s-5 (45 mg, 0.021 mmol, 1.0 equiv.) was dissolved in CH2Cl2. EDC, HOBt,and N-hydroxysuccinimide were added sequentially, followed by DIPEA, andthe reaction was allowed to stir at room temperature for 6 hours, afterwhich conversion was observed via LCMS. The reaction was concentrated,and a mixture of 95% TFA, 2.5% PBS, 2.5% TIPS was added. The reactionwas allowed to stir for 30 minutes, after which it was concentrated. 1mL of PBS and 1.5 mL of a saturated sodium bicarbonate solution wasadded immediately to the flask, followed by cp33 peptide (16 mg, 0.007mmol, 0.3 equiv.). The reaction was allowed to stir at room temperaturefor 12 hours, and subsequently purified by HPLC (C18 reverse phase, 5mL/min, 30%-42% MeCN in H₂O over 66 minutes). The fractions containingproduct were collected and lyophilized to give a white powder (1.2 mg,11.2%).

(S)-di-tert-butyl2-(3-((S)-6-(4-(1-(9H-fluoren-9-yl)-3,31-dioxo-2,7,10,13,16,19,22,25,28,35,38,41,44,47,50,53,56-heptadecaoxa-4,32-diazaheptapentacontan-57-yl)-1H-1,2,3-triazol-1-yl)-1-(tert-butoxy)-1-oxohexan-2-yl)ureido)pentanedioate(s-6)

s-2 (90 mg, 0.135 mmol, 1 equiv.) was dissolved in 1 mL of dry CH₂Cl₂ ina flame dried flask, and EDC.HCl (29 mg, 0.15 mmol, 1.1 equiv.) andHOBt.H2O (23 mg, 0.15 mmol, 1.1 equiv) were sequentially added, followedby a solution of Fmoc-N-amido-dPEG8-acid (Quanta Biodesign Ltd. 138 mg,0.15 mmol, 1.1 equiv.) in 1 mL of dry CH₂C12 and DIPEA (28 uL, 0.162mmol, 1.2 equiv.). The reaction was allowed to stir for 2.5 hours, afterwhich 1 mL of MeOH was added. The reaction was then concentrated underreduced pressure and chromatographed (silica gel, 1×15 cm, 2.5% MeOH inCHCl₃, then 5% MeOH in CHCl₃, then 10% MeOH in CHCl₃) to yield s-6 as aclear oil (149 mg, 70%). IR (thin film) 3332 (br), 2868 (m), 1727 (m),1672 (w), 1547 (m), 1451 (w), 1367 (w), 1251 (m), 1149 (s), 1110 (s)cm-1. 1HNMR (500 MHz, CDCl3) d 7.75 (d, J=7.5 Hz, 2H) 7.62 (s, 1H), 7.61(d, J=7.5 Hz, 2H), 7.39 (t, J=7.5 Hz, 2H), 7.31 (t, J=7.5 Hz, 2H), 6.63(s, 1H), 5.42 (s, 1H), 5.24 (d, J=8 Hz, 1H), 5.15 (d, J=8 Hz, 1H), 4.67(dd, J=10 Hz, 25 Hz, 2H), 4.40 (d, J=7 Hz, 2H), 4.37-4.27 (m, 4H), 4.22(t, J=7 Hz, 1H), 3.72 (t, J=6 Hz, 2H), 3.68-3.55 (m, 66H), 3.54 (t, J=5Hz, 2H), 3.43 (dd, J=5.5, 12 Hz, 2H), 3.39 (dd, J=5.5, 12 Hz, 2H), 2.46(t, J=6 Hz, 2H), 2.37-2.24 (m, 2H) 2.10-2.03 (m, 2H), 1.95-1.76 (m, 5H),1.64-1.57 (m, 1H), 1.46 (s, 9H), 1.43 (s, 18H), 1.39-1.29 (m, 2H).13CNMR (100 MHz, CDCl3) d 172.4, 172.1, 172.1, 171.4, 156.8, 145.1,144.0, 141.3, 127.7, 127.0, 125.1, 122.9, 120.0, 81.9, 81.9, 80.6, 70.6,70.6, 70.6, 70.5, 70.4, 70.4, 70.3, 70.3. 70.1, 69.9, 69.6, 67.3, 66.6,64.6, 53.1, 53.0, 49.9, 47.3, 41.0, 39.2, 37.0, 32.4, 31.7, 29.6, 28.3,28.1, 28.1, 28.0, 21.8. HRMS (ES+) calc'd for C₇₇H₁₂₇N₇O₂₆ (M+H) m/z1566.8904. Found 1566.8892.

(S)-di-tert-butyl2-(3-((S)-6-(4-(53-amino-27-oxo-2,5,8,11,14,17,20,23,30,33,36,39,42,45,48,51-hexadecaoxa-26-azatripentacontyl)-1H-1,2,3-triazol-1-yl)-1-(tert-butoxy)-1-oxohexan-2-yl)ureido)pentanedioate(s-7)

s-6 (135 mg) was dissolved in 3 mL of Et₂NH and 3 mL of CH₂Cl₂. Thereaction was allowed to stir at room temperature for 3 hours, afterwhich it was concentrated under reduced pressure and chromatographed(silica gel, 2×15 cm, 2.5% MeOH in CHCl₃, then 5% MeOH in CHCl₃, then10% MeOH in CHCl₃, then 20% MeOH in CHCl₃, then 20% MeOH in CHCl₃+2.5%Et₃N) to yield s-7 as a clear oil (86 mg, 75%). IR (thin film). 1HNMR(400 MHz, MeOD) d 8.01 (s, 1H), 4.64 (s, 2H), 4.42 (t, J=7.2 Hz, 3H),4.20-4.17 (m, 1H), 4.15-4.11 (m, 1H), 3.77-3.63 (m, 72H), 3.54 (t, J=5.6Hz, 2H), 3.37 (t, J=5.6 Hz, 2H), 3.14 (t, J=5.2 Hz, 2H), 3.00 (dd,J=7.2, 15.2 Hz, 1H), 2.61 (t, J=6 Hz, 1H), 2.48 (t, J=6 Hz, 2H),2.38-2.25 (m, 2H), 2.08-2.00 (m, 1H), 1.98-1.90 (m, 2H), 1.85-1.74 (m,2H), 1.70-1.60 (m, 1H), 1.48 (s, 9H), 1.44 (s, 18H), 1.40-1.36 (m, 2H),1.29 (t, J=7.2 Hz, 2H). 13CNMR (100 MHz, MeOD) d 173.9, 173.8, 173.7,173.5, 159.9, 146.0, 125.1, 82.8, 82.7, 81.8, 71.6, 71.5, 71.5, 71.4,71.4, 71.3, 71.3, 71.2, 71.2, 71.1, 71.1, 70.8, 70.7, 70.6, 68.5, 68.3,67.6, 65.0, 54.7, 54.2, 52.2, 51.1, 43.7, 40.8, 40.5, 37.5, 35.7, 32.9,32.5, 30.9, 29.0, 28.4, 28.4, 23.5. HRMS (ES+) calc'd for C₆₂H₁₁₇N₇O₂₄(M+H) m/z 1344.8223. Found 1344.8251.

s-8

s-3 (8 mg, 0.023 mmol, 1.0 equiv.) was dissolved in CH₂Cl₂ (1 mL). Tothis solution were added EDC.HCl (15 mg, 0.076 mmol, 3.3 equiv.),HOBt.H₂O (12 mg, 0.076 mmol, 3.3 equiv.), and a solution of s-7 (102 mg,0.076 mmol, 3.3 equiv.) in CH₂Cl₂ (4 mL). Pyridine (5.5 mg, 5.7 μL, 0.7mmol, 3.0 equiv.), was added, and the reaction was allowed to proceed atroom temperature for 16 hours, after which it was concentrated andpartially purified (1×15 cm, silica gel, 10% MeOH in CHCl₃). Thefractions containing product was concentrated and immediately taken upwith MeOH (1 mL). 1 M LiOH in H₂O (92 μL, 0.092 mmol, 4 equiv.) wasadded to the solution, and the reaction was allowed to stir at roomtemperature for 3 hours, after which 1 M LiOH in H₂O (92 μL, 0.092 mmol,4 equiv.) was added. The reaction was allowed to stir at roomtemperature for an additional 2 hours, after which it was neutralizedwith 1 M HCl in H₂O (138 mL, 0.138 mmol, 6.0 equiv.). The reaction wasconcentrated, taken up with 80% MeCN in H₂O, and purified by HPLC(reverse phase, C18, 5 mL/min, 50%-75% MeCN in H₂O over 40 minutes). Theproduct was isolated and lyophilized to yield s-8 as a white powder (8mg,). IR (thin film) 2873 (m), 1729 (m), 1666 (s), 1555 (w), 1456 (w),1368 (w), 1140 (s) 950 (w), 846 (w) cm-1. 1HNMR (500 MHz, MeOD) d 8.72(t, J=5.2 Hz, 1H), 8.51 (s), 3H), 8.42 (s, 1H), 7.99 (s, 2H), 7.90 (s),3H), 4.62 (s, 4H), 4.41 (t, J=7 Hz, 4H), 4.18 (dd, J=5, 7.2 Hz, 2H),4.13 (dd, J=5, 6 Hz, 2H), 3.71-3.69 (m, 10H), 3.66-3.57 (m, 118H), 3.52(t, J=5.5 Hz, 411), 3.36-3.34 (m, 8H), 2.84 (t, J=6 Hz, 2H), 2.44 (t,J=6.2 Hz, 4H), 2.37 (t, J=7.2 Hz, 2H), 2.33-2.28 (m, 4H), 2.06-2.00 (m,2H), 1.96-1.90 (m, 4H), 1.83-1.77 (m, 6H), 1.75-1.68 (m, 2H), 1.67-1.63(m, 2H), 1.46 (s, 18H), 1.44 (s, 18H), 1.43 (s, 18H), 1.39-1.34 (m, 4H).13CNMR (125 MHz, MeOD) d 177.2, 174.0, 173.7, 173.7, 173.5, 167.9,159.9, 150.2, 146.0, 138.8, 138.0, 127.4, 125.1, 122.8, 121.8, 82.8,82.7, 81.8, 79.5, 71.5, 71.5, 71.5, 71.4, 71.4, 71.4, 71.3, 71.3, 71.3,70.7, 70.6, 70.5, 68.3, 65.0, 54.7, 54.2, 51.1, 41.3, 40.4, 37.5, 34.6,32.9, 32.5, 30.8, 29.8, 29.0, 28.4, 28.3, 28.3, 26.1, 25.5, 25.3, 23.5.HRMS (ES+) calc'd for C₁₃₉H₂₄₅N₁₇O₅₂ (M+H) m/z 2985.7150. Found (M+2H)m/z 1493.3458.

s-9

s-8 (22 mg, 0.0074 mmol, 1.0 equiv.) was dissolved in 1 mL of DMF. Tothis solution was added EDC (14.2 mg, 0.074 mmol, 10.0 equiv.), HOBt(11.3 mg, 0.074 mmol, 10.0 equiv.), and NHS (8.5 mg, 0.074 mmol, 10.0equiv.) sequentially, followed by DIPEA (13.0 μL, 0.074 mmol, 10.0equiv.). The reaction was allowed to stir at room temperature for 13hours, after which the DMF was removed by passing a steady stream of N2over the reaction. A mixture of 97.5% TFA/2.5% TIPS was added to theflask, and the reaction was allowed to stir for 1 hour, after which theTFA was removed under reduced pressure. The crude reaction mixture waspurified by HPLC (Sunfire™ Prep C18 column (10×150 mm) using a 50% MeCNto 80% MeCN in H₂O gradient over 66 min at 5 mL/min) to give s-9 as awhite powder (2.5 mg), which was immediately used for the next step.

cp33-Bis(Peg16-Urea)

s-9 (2.5 mg, 0.9 μmol, 1.0 equiv.) was dissolved in 2 mL of PBS and 1 mLof saturated sodium bicarbonate in water. cp33 peptide (2.5 mg, 0.11mmol, 1.2 equiv.) was added, and the reaction was allowed to stir atroom temperature for 4 hours, after which it was injected directly ontothe HPLC and purified (Sunfire™ Prep C18 column (10×150 mm) using a 30%MeCN to 42% MeCN in H₂O gradient over 39 min at 5 mL/min). The fractionscontaining the product were isolated and lyophilized to give a whitepowder (1.4 mg, 32.5%).

benzyl 6-(6-((tert-butoxycarbonyl)amino)hexanamido)hexanoate (s-9)

6-((tert-butoxycarbonyl)amino)hexanoic acid (2.58 g, 11.15 mmol, 1.1equiv.) was dissolved in 50 mL of DCM. To the solution were addedEDC.HCl (2.14 g, 11.15 mmol, 1.1 equiv.), HOBt.H₂O (1.71 g, 11.15 mmol,1.1 equiv.), and a solution of benzyl 6-aminohexanoate (2.24 g, 10.14mmol, 1.0 equiv.) in CH₂Cl₂ (50 mL). DIPEA (2.0 mL, 11.15 mmol, 1.1equiv.) was added, and the solution was allowed to stir at roomtemperature for 90 min, after which the reaction was washed with 10%Citric Acid (100 mL), saturated NaHCO₃ (100 mL), and brine (100 mL). Theorganic layer was collected, dried with MgSO₄, and concentrated to gives-9 (3.17 g, 73% crude) as a white powder, which was carried on withoutany further purification.

benzyl 6-(6-aminohexanamido)hexanoate (s-10)

s-9 (3.1 g) was dissolved in 15 mL of TFA and stirred at roomtemperature for 1 hour, after which the TFA was partially removed underreduced pressure. The resulting oil was washed with 150 mL of diethylether, and the ether was carefully decanted into 50 mL centrifuge tubes.The centrifuge tubes were spun down at 3.0 rcf for 10 minutes, resultingin the product settling to the bottom, and the ethereal layer wascarefully removed. The products were combined with methanol,concentrated down under reduced pressure, and azeotroped with deuteratedchloroform to yield s-10 as a green oil (2.27 g, 92% crude), which wascarried on without purification.

benzyl1-(9H-fluoren-9-yl)-3,10,17-trioxo-2-oxa-4,11,18-triazatetracosan-24-oate(s-11)

6-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)hexanoic acid (2.39 g, 6.78mmol, 1 equiv.) was dissolved in CH₂Cl₂ (35 mL). To this solution wasadded EDC.HCl (1.56 g, 8.14 mmol, 1.2 equiv.), and HOBt.H₂O (1.25 g,8.14 mmol, 1.2 equiv.). In a separate flask, s-10 (2.27 g, 6.78 mmol, 1equiv.) was dissolved in CH₂Cl₂ (40 mL), and DIPEA (3 mL, 17 mmol, 2.5equiv.) was added to neutralize leftover TFA. This solution was added tothe original reaction, and the flask was washed with an additional 10 mLof CH₂Cl₂, which was also added to the original reaction. The reactionmixture was stirred for 75 minutes at room temperature, after which itwas washed with 10% Citric acid (100 mL), saturated sodium bicarbonate(100 mL), and saturated brine (100 mL). The organic layer was collected,dried with MgSO₄, concentrated under reduced pressure, and partiallypurified (silica gel, 3×25 cm, 5% MeOH in CHCl₃, then 10% MeOH in CHCl₃)to remove coupling reagents. The fractions containing the product werecollected and chromatographed a second time (50% MeCN in CHCl₃+2.5%DIPEA) to yield a white solid, which was chromatographed a third time(5% MeOH in CHCl₃, then 10% MeOH in CHCl₃) to yield s-11 as a pure,white solid (1.5 g, 33%, 3 steps). IR (thin film) 3306 (br), 2936 (m),2861 (w), 1719 (s), 1647 (s), 1544 (s), 1450 (m), 1254 (s), 1160 (m),741 (m) cm-1. 1HNMR (400 MHz, MeOD) d 7.80 (d, J=8 Hz, 2H), 7.64 (d, J=8Hz, 2H), 7.39 (t, J=8 Hz, 2H), 7.35-7.32 (m, 5H), 7.30 (t, J=8 Hz, 2H),5.10 (s, 2H), 4.33 (d, J=6.4 Hz, 2H), 4.21 (t, J=7.2 Hz, 1H), 3.16-3.08(m, 6H), 2.36 (t, J=7.2 Hz, 2H), 2.19-2.13 (m, 4H), 1.65-1.58 (m, 6H),1.53-1.46 (m, 6H), 1.36-1.30 (m, 6H). 13CNMR (100 MHz, MeOD) d 176.0,175.0, 158.9, 145.3, 142.6, 137.7, 129.5, 129.2, 128.8, 128.1, 126.2,120.9, 67.6, 67.1, 41.6, 40.2, 40.1, 37.0, 37.0, 34.9, 30.6, 30.0, 27.5,27.4, 27.4, 26.7, 25.7. HRMS (ES+) calc'd for C₄₀H₅₁N₃O₆ (M+H) m/z670.3850. Found 670.3847.

1-(9H-fluoren-9-yl)-3,10,17-trioxo-2-oxa-4,11,18-triazatetracosan-24-oicacid (s-12)

s-11 was taken up with 90% iPrOH/10% MeOH (30 mL) and added to a slurryof 10% Pd/C in 10 mL of 90% iPrOH/10% MeOH. 40 mL of 90% iPrOH/10% MeOHwas used to add the remaining starting material, and the flask waspurged with N₂ for 15 minutes. H₂ gas was then bubbled through theslurry for 5 minutes, and the reaction was allowed to stir for 2 hoursunder H₂ atmosphere at room temperature, after which it was filteredwith celite and chromatographed (3×25 cm silica gel, 10% MeOH in CHCl₃)to yield s-12 as a white solid (580 mg, 45%). IR (thin film) 3312 (br),2935 (m), 2861 (w), 1705 (s), 1647 (s), 1546 (s), 1450 (w), 1255 (m)cm-1. 1HNMR (400 MHz, MeOD), 7.97 (s, 2H), 7.83 (d, J=8 Hz, 2H), 7.67(d, J=8 Hz, 2H), 7.62 (t, J=8 Hz, 2H), 7.35-7.32 (t, J=8 Hz, 2H), 7.13(s, 1H), 4.37 (d, J=6.4 Hz, 2H), 4.22 (t, J=7.2 Hz, 1H), 3.20-3.12 (m,6H), 2.31 (t, J=7.2 Hz, 2H), 2.22-2.16 (m, 4H), 1.66-1.59 (m, 6H),1.56-1.49 (m, 6H), 1.42-1.33 (m, 6H). 13CNMR (100 MHz, MeOD) d 177.6,176.1, 158.9, 145.4, 142.6, 128.8, 128.2, 126.2, 121.0, 67.6, 41.6,40.2, 37.0, 37.0, 30.6, 30.1, 27.6, 27.5, 27.4, 26.8, 26.7, 25.8. HRMS(ES+) calc'd for C₃₃H₄₅N₃O₆ (M+H) m/z 580.3381. Found 580.3377.

benzyl 6-(6-(6-aminohexanamido)hexanamido)hexanoate (s-13)

s-12 (1.17 g) was dissolved in a mixture of 1:1 Et₂NH/CH₂Cl₂ (37 mL),and the reaction was stirred at room temperature for 8 hours, afterwhich it was concentrated under reduced pressure and chromatographed(3×25 cm silica gel, 20% MeOH in CHCl₃, then 20% MeOH in CHCl₃+2.5%Et₃N) to yield s-13 as a white solid (630 mg, 81%). IR (thin film) 3221(w), 3277 (w), 2941 (m), 2862 (w), 1727 (m), 1633 (s), 1542 (m), 1477(w), 1262 (w), 1184 (w) cm-1. 1HNMR (400 MHz, CDCl3) d 7.38-7.30 (m,5H), 5.89 (s, 1H), 5.67 (s, 1H), 5.11 (s, 2H), 3.26-3.20 (m, 6H), 2.27(t, J=7.2, 2H), 2.19-2.14 (m, 4H), 1.69-1.62 (m, 6H), 1.55-1.48 (m, 6H),1.40-1.32 (m, 6H). 13CNMR (100 MHz, CDCl3) d 173.5, 173.0, 172.9, 136.0,128.4, 128.3, 128.2, 66.2, 41.7, 39.2, 39.1, 36.6, 36.5, 34.1, 32.5,29.3, 29.2, 26.4, 25.4, 25.1, 24.5. HRMS (ES+) calc'd for C₂₅H₄₁N₃O₄(M+H) m/z 448.3170. Found 448.3150.

6,6′-((6,6′-((6,6′-((5-azidoisophthaloyl)bis(azanediyl))bis(hexanoyl))bis(azanediyl))bis(hexanoyl))bis(azanediyl))dihexanoicacid (s-14)

A mixture of azidoisophthalic acid (100 mg, 0.483 mmol, 1 equiv.) in 5mL of dry CH₂Cl₂ was prepared in a flame dried flask. To this mixturewas sequentially added EDC.HCl (233 mg, 1.21 mmol, 2.5 equiv.), HOBt.H2O(183 mg, 1.21 mmol, 2.5 equiv.), s-13 (476 mg, 1.06 mmol, 2.2 equiv.),and pyridine (0.117 mL, 1.45 mmol, 3 equiv.). The reaction was allowedto stir at room temperature for 18 hours, after which it was directlyloaded onto a silica gel column and partially purified to remove thecoupling agents (silica gel, 2×15 cm, 10% MeOH in CHCl₃). The crudematerial was dissolved in 5 mL of MeOH and 5 mL of THF. A 1 M solutionof LiOH in H₂O was added (1.9 mL, 4 equiv.), and the reaction wasallowed to stir at room temperature for 24 hours. A solution of 1 M HClwas added (0.95 mL, 2 equiv.), and the reaction was concentrated underreduced pressure. The crude mixture was then chromatographed (silicagel, 2×15 cm, 20% MeOH in CHCl₃, then 20% MeOH in CHCl₃+1% TFA) andfractions containing the product were collected and concentrated underreduced pressure to obtain an light brown oil. 50 mL of 95% diethylether/5% MeOH was then added to the oil. The ether/MeOH mixture was thendecanted, and the oil dried under reduced pressure to obtain s-14 as awhite solid (383 mg, 89%, 2 steps). IR (thin film) 3310 (br), 2937 (m),2865 (w), 2112 (m), 1650 (s), 1553 (m), 1439 (w), 1202 (m), 1139 (m)cm-1. 1HNMR (400 MHz, MeOD) d 8.05 (s, 1H), 7.65 (s, 2H), 3.39 (t, J=5.6Hz, 4H), 3.15 (dd, J=3.2, 6.6 Hz, 8H), 2.29 (t, 7.2 Hz, 4H), 2.22-2.15(m, 8H), 1.68-1.58 (m, 16H), 1.51-1.47 (m, 8H), 1.42-1.32 (m, 1211).13CNMR (100 MHz, MeOD) d 177.4, 176.0, 168.3, 138.1, 123.6, 121.5, 41.0,40.2, 37.0, 34.8, 30.1, 30.1, 27.6, 27.5, 26.7, 25.7. HRMS (ES+) calc'dfor C₄₄H₇₁N₉O₁₀ (M+H) m/z 886.5397. Found 886.5408.

(S)-di-tert-butyl2-(3-((S)-6-(4-(aminomethyl)-1H-1,2,3-triazol-1-yl)-1-(tert-butoxy)-1-oxohexan-2-yl)ureido)pentanedioate(s-15)

A mixture of s-1 (180 mg, 0.35 mmol, 1 equiv.) and propargylamine (117uL, 1.75 mmol, 5 equiv.) was dissolved in a mixture of H₂O (1.25 mL) andt-butanol (1.25 mL) in a 5 mL microwave reaction tube. To the mixturewas added 0.1 M sodium ascorbate (0.7 mL, 0.2 equiv.) and 0.1 Mcopper(II) sulfate (0.14 mL, 0.04 equiv.). The tube was capped, andsubject to microwave irradiation for 2.5 minutes at 110° C. The reactionwas then concentrated under reduced pressure at 60° C., andchromatographed (silica gel, 1×15 cm, 10% MeOH in CHCl₃, then 10% MeOHin CHCl₃+2.5% Et₃N) to obtain s-15 as a pale, green oil (152 mg, 77%).IR (thin film) 2978 (m), 2933 (w), 1730 (s), 1647 (m), 1559 (m), 1456(w), 1368 (m), 1255 (w), 1154 (s) cm-1. 1HNMR (400 MHz, MeOD) d 7.90 (s,1H), 4.90 (t, J=7.2 Hz, 2H), 4.20-4.16 (m, 1H), 4.14-4.12 (m, 1H), 3.96(s, 2H), 2.36-2.26 (m, 2H), 2.08-2.00 (m, 1H), 1.95-1.89 (m, 2H),1.84-1.73 (m, 2H), 1.69-1.59 (m, 1H), 1.46 (s, 9H), 1.44 (s, 18H),1.39-1.31 (m, 2H). 13CNMR (100 MHz, MeOD) d 173.7, 173.7, 173.5, 159.9,147.7, 123.8, 82.8, 82.7, 81.8, 54.2, 51.1, 37.1, 32.9, 32.5, 30.8,29.0, 28.4, 28.3, 23.5. HRMS (ES+) calc'd for C₂₇H₄₈N₆O₇ (M+H) m/z568.3657. Found 568.3637.

(S)-di-tert-butyl2-(3-((S)-6-(4-(1-(9H-fluoren-9-yl)-3,10,17,24-tetraoxo-2-oxa-4,11,18,25-tetraazahexacosan-26-yl)-1H-1,2,3-triazol-1-yl)-1-(tert-butoxy)-1-oxohexan-2-yl)ureido)pentanedioate(s-16)

A mixture of 6-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)hexanoic acid(156 mg, 0.267 mml, 1 equiv.) was prepared in 1.5 mL of dry CH₂Cl₂. Tothis slurry was added EDC.HCl (56 mg, 0.294 mmol, 1.1 equiv.) andHOBt.H₂O (45 mg, 0.294 mmol, 1.1 equiv.). A solution of s-16 (152 mg,0.267 mmol, 1 equiv.) in 3 mL of dry CH₂Cl₂, followed by DIPEA (32 uL,0.294 mmol, 1.1 equiv.), was added to this slurry. The reaction wasallowed to stir at room temperature for 2 hours, after which EDC-HCl (28mg, 0.247 mmol, 0.5 equiv.), HOBt-H₂O (23 mg, 0.247 mmol, 0.5 equiv.),and DIPEA (32 uL, 0.294 mmol, 1.1 equiv.) were added. The reaction wasallowed to stir at room temperature for an additional 2 hours, afterwhich the reaction was concentrated down under reduced pressure andchromatographed (silica gel, 2×15 cm, 10% MeOH in CHCl₃) to give s-16 asa viscous, pale green oil (235 mg, 78%). IR (thin film) 2933 (m), 2862(w), 2413 (br), 1729 (s), 1630 (s), 1453 (s), 1367 (m), 1251 (w), 1153(s), 742 (w) cm-1. 1HNMR (400 MHz, MeOD) d 7.84 (s, 1H), 7.80 (d, J=7.2Hz, 2H), 7.64 (d, J=7.2 Hz, 2H), 7.39 (t, J=8 Hz, 2H), 4.40 (d, J=2 Hz,2H), 4.37 (t, J=6.8 Hz, 2H), 4.33 (d, J=6.8 Hz, 2H), 4.21-4.17 (m, 2H),4.13 (dd, J=5.2, 8 Hz, 1H), 3.17-3.08 (m, 6H), 2.34-2.29 (m, 2H),2.23-2.13 (m, 6H), 2.08-2.00 (m, 1H), 1.94-1.86 (m, 2H), 1.84-1.73 (m,2H), 1.64-1.57 (m, 7H), 1.52-1.43 (m, 5H), 1.46 (s, 9H), 1.43 (s, 18H),1.39-1.29 (m, 9H). 13CNMR (100 MHz, MeOD) d 176.0, 175.9, 173.7, 173.6,173.4, 159.9, 158.8, 146.2, 145.3, 142.6, 128.8, 128.1, 126.2, 124.1,120.9, 82.8, 82.6, 81.7, 67.6, 54.6, 54.1, 51.3, 41.6, 40.2, 37.0, 37.0,36.7, 35.6, 32.9, 30.8, 30.6, 30.1, 29.0, 28.4, 28.3, 27.5, 27.4, 26.7,26.7, 26.5, 23.4. HRMS (ES+) calc'd for C₆₀H₉₁N₉O₁₂ (M+H) m/z 1130.6860.Found 1130.6848.

(S)-di-tert-butyl2-(3-((S)-6-(4-((6-(6-(6-aminohexanamido)hexanamido)hexanamido)methyl)-1H-1,2,3-triazol-1-yl)-1-(tert-butoxy)-1-oxohexan-2-yl)ureido)pentanedioate(s-17)

s-16 (195 mg) was dissolved in 2.5 mL of Et₂NH and 2.5 mL of CH₂Cl₂. Thereaction was allowed to stir at room temperature for 26 hours, afterwhich it was concentrated under reduced pressure and chromatographed(silica gel, 2×15 cm, 20% MeOH in CHCl₃, then 20% MeOH in CHCl₃+2.5%Et₃N) to obtain s-17 as a sticky, pale yellow solid (137 mg, 90%). IR(thin film) 3271 (br), 2934 (m), 2864 (w), 1731 (s), 1643 (s), 1556 (s),1457 (w), 1367 (m), 1256 (w), 1154 (s) cm-1. 1HNMR (400 MHz, MeOD) d7.99 (s, 1H), 7.88 (s, 1H), 6.40 (dd, J=12, 14 Hz, 1H), 4.42 (s, 2H),4.41 (t, J=9 Hz, 2H), 4.21-4.11 (m, 2H), 3.22-3.14 (m, 4H), 2.93 (t, J=8Hz, 2H), 2.35-2.30 (m, 2H), 2.25-2.16 (m, 6H), 2.08-2.01 (m, 1H),1.95-1.90 (m, 1H), 1.83-1.74 (m, 2H), 1.71-1.58 (m, 9H), 1.55-148 (m,4H), 1.48 (s, 9H), 1.45 (s, 18H), 1.42-1.29 (m, 10H). 13CNMR (100 MHz,MeOD) d 176.0, 175.7, 173.8, 173.7, 173.5, 159.9, 146.3, 124.2, 82.8,82.7, 81.8, 81.7, 40.6, 40.2, 40.2, 37.0, 36.8, 36.6, 35.6, 32.9, 32.5,30.8, 30.6, 30.2, 29.0, 28.4, 28.3, 27.6, 27.0, 26.8, 26.5, 26.4, 23.5.HRMS (ES+) calc'd for C₃₄H₈₁N₉O₁₀ (M+H) m/z 908.6179. Found 908.6165.

s-18

To a solution of s-14 (150 mg, 0.169 mmol, 1.0 equiv.) in CH₂Cl₂ (1.5mL) was added EDC.HCl (81 mg, 0.423 mmol, 2.5 equiv.), HOBt.H₂O (65 mg,0.423 mmol, 2.5 equiv.), followed by a solution of s-15 (212 mg, 0.373mmol, 2.2 equiv.) in CH₂Cl₂ (3.5 mL). Pyridine (82 □L, 0.507 mmol, 3equiv.) was added to the resulting solution, and the reaction wasallowed to stir at room temperature for 20 hours, after which it wasconcentrated under reduced pressure and partially purified by silica gelchromatography (1×15 cm, silica gel, 5% MeOH in CHCl₃, then 10% MeOH inCHCl₃) to yield a dark red oil (190 mg, 57% crude) that was carried ontothe next step.

s-19

Partially pure s-18 (100 mg, 0.05 mmol, 1.0 equiv.) was dissolved in H₂O(1.25 mL) and t-BuOH (1.25 mL) in a 5 mL microwave vial. To thissolution was added 6-heptynoic acid (6.4 μL, 0.05 mmol, 1.0 equiv.), 0.1M Sodium Ascorbate (0.5 mL, 0.05 mmol, 1.0 equiv.), and 0.1 M copper(II)sulfate (0.25 mL, 0.025 mmol, 0.5 equiv.). The vial was sealed andsubjected to microwave irradiation for 1 hour at 110° C., after whichthe reaction was concentrated and purified by HPLC (Sunfire™ Prep C18column (10×150 mm) using a 50% MeCN to 80% MeCN in H₂O gradient over 66min at 5 mL/min). The product was isolated and lyophilized to yield awhite powder (11 mg, 11%). IR (thin film) 2938 (w), 1634 (s), 1553 (m),1461 (m), 1369 (w), 1141 (s), 975 (w), 841 (w), 800 (w), 722 (w) cm-1.1HNMR (400 MHz, MeOD) d 8.78 (t, J=5.4 Hz, 1H), 8.48 (s, 1H), 8.45 (d,J=1.6 Hz, 2H), 8.36 (s, 1H), 7.88 (s, 2H), 4.41-4.37 (m, 8H), 4.18 (dd,J=5, 8.8 Hz, 2H), 4.12 (dd, J=5.2, 8 Hz, 2H), 3.67-3.64 (m, 1H), 3.43(t, J=6.8 Hz, 4H), 3.14 (t, J=6.4 Hz, 8H), 2.84 (t, J=7 Hz, 2H), 2.37(t, J=7.2 Hz, 2H), 2.34-2.29 (m, 4H), 2.26-2.13 (m, 12H), 2.08-1.99 (m,2H), 1.95-1.87 (m, 4H), 1.84-1.71 (m, 6H), 1.69-1.54 (m, 20H), 1.50-1.42(m, 10H), 1.46 (s, 18H), 1.44 (s, 18H), 1.43 (s, 18H), 1.38-1.26 (m,12H). 13CNMR (100 MHz, MeOD) d 176.0, 173.7, 173.7, 173.5, 167.9, 159.9,138.2, 122.6, 82.8, 82.7, 81.7, 54.6, 54.2, 40.2, 37.0, 32.9, 32.5,30.1, 29.0, 28.4, 28.3, 27.6, 26.7, 23.4. HRMS (ES+) calc'd forC₁₀₅H₁₇₃N₂₁O₂₄ (M+H) m/z 2113.3062. Found (M+2H) 1057.1462.

s-20

Partially pure s-19 (35 mg, 0.018 mmol, 1.0 equiv.) was dissolved inTHF. To this solution was added the N-hydroxysuccinimidyl ester oftrifluoroacetic acid (19 mg, 0.09 mmol, 5.0 equiv.) and pyridine (10 mg,0.125 mmol, 7.0 equiv.). The reaction was allowed to stir at roomtemperature for 1.5 hours, after which it was quenched with 0.5 mL ofPBS and concentrated under reduced pressure. To the resulting mixturewas added 2 mL of 95% TFA/5% TIPS, and the reaction was allowed to stirat room temperature for 1 hour, after which the TFA was removed underreduced pressure. The reaction mixture was purified by HPLC (Sunfire™Prep C18 column (10×150 mm) using a 0% MeCN to 80% MeCN in 1120 gradientover 51 min at 5 mL/min) and fractions containing the product wasisolated and lyophilized to give s-20 (1.9 mg), which was usedimmediately in the next reaction.

cp33-Bis(Ac3-Urea)

s-20 was dissolved in 975 □L of PBS and 325 □L of saturated sodiumbicarbonate in water. cp33 peptide (2.4 mg, 0.0011 mmol, 1.1 equiv.) wasadded, and the reaction was allowed to stir at room temperature for 7hours, after which the reaction mixture was loaded directly onto theHPLC and purified (Sunfire™ Prep C18 column (10×150 mm) using a 0% MeCNto 80% MeCN in H2O gradient over 51 min at 5 mL/min). The fractionscontaining the product was collected and lyophilized to give a whitepowder (1.1 mg, 25.6%).

s-21

A solution of s-14 (190 mg, 0.214 mmol, 1.0 equiv.) was prepared inCH₂Cl₂ (5 mL). EDC.HCl (103 mg, 0.535 mmol, 2.5 equiv.) and HOBt.H₂O (82mg, 0.535 mmol, 2.5 equiv.) were added, followed by a solution of s-17(428 mg, 0.472 mmol, 2.2 equiv.) in CH₂Cl₂ (5 mL). Pyridine (52 μL,0.642 mmol, 3.0 equiv.) was added, and the reaction was allowed to stirat room temperature for 24 hr, after which it was concentrated underreduced pressure and chromatographed (Silica gel, 1×15 cm, 10% MeOH inCHCl₃, then 15% MeOH in CHCl₃, then 20% MeOH in CHCl₃) to give apartially pure, pale yellow solid (250 mg, 44% crude).

s-22

s-21 (50 mg, 0.019 mmol, 1.0 equiv.) was dissolved in H₂O (0.5 mL) andt-BuOH (0.5 mL) in a 2 mL microwave vial. To this solution was added6-heptynoic acid (2.5 μL, 0.019 mmol, 1.0 equiv.), 0.1 M SodiumAscorbate (0.2 mL, 1.0 equiv.), and 0.1 M copper(II) sulfate (0.1 mL,0.5 equiv.). The vial was sealed and subjected to microwave irradiationfor 1 hour at 110° C., after which the reaction was concentrated andchromatographed (1×15 cm, silica gel, 20% MeOH in CHCl₃, then 20% MeOHin CHCl₃+1% TFA). The fractions containing the product wereconcentrated, washed with diethyl ether (50 mL), and azeotroped withCDCl₃ to give a partially pure green solid (27 mg) that was carriedforward to the next step.

s-23

Partially pure s-22 (37 mg, 0.013 mmol, 1.0 equiv.) was dissolved in DMF(0.5 mL). EDC (7.7 mg, 0.04 mmol, 3.0 equiv.), HOBt (6.2 mg, 0.04 mmol,3.0 equiv.), and N-hydroxysuccinimide (4.6 mg, 0.04 mmol, 3.0 equiv.)were added sequentially, followed by DIPEA (7.1 μL, 0.04 mmol, 3.0equiv.). The reaction was allowed to stir for 1 hour, after which EDC(7.7 mg, 0.04 mmol, 3.0 equiv.) and DIPEA (7.1 μL, 0.04 mmol, 3.0equiv.) were added. The reaction was allowed to stir for another 2hours, after which the DMF was removed by passing a steady stream of N2over the reaction. 1 mL of a mixture of 98% TFA/2% TIPS was added to theflask, and the reaction was allowed to stir at room temperature for 30minutes, after which the TFA was removed under reduced pressure. Thereaction was purified by HPLC (Sunfire™ Prep C18 column (10×150 mm)using a 0% MeCN to 80% MeCN in H₂O gradient over 51 min at 5 mL/min).Fractions containing the product were collected and lyophilized to gives-23 as a white powder (2.5 mg), which was used immediately in the nextstep.

cp33-Bis(Ac6-Urea)

s-23 (2.5 mg, 0.86 μmol, 1.0 equiv.) was dissolved in 0.9 mL of PBS and0.4 mL of a 7.5% solution of sodium bicarbonate in water. cp33 (2 mg,0.65 μmol, 0.75 equiv.) was added, and the reaction was allowed to stirat room temperature for 1 hour, after which 1 mL of DMF was added. Thereaction was allowed to stir for an additional 6 hours at roomtemperature, after which the reaction mixture was injected directly ontothe HPLC and purified (Sunfire™ Prep C18 column (10×150 mm) using a 10%MeCN to 65% MeCN in H2O gradient over 66 min at 5 mL/min). Fractionscontaining the product were collected and lyophilized to givecp33-Bis(Ac6-Urea) as a white powder (0.2 mg, 4.5%).

s-22

3,6,9,12,15,18,21,24-octaoxaheptacos-26-yn-1-amine (3 mg, 0.007 mmol,1.0 equiv.) was added to a 2 mL microwave vial, followed by a solutionof s-20 (20 mg, 0.007 mmol, 1.0 equiv.) in H₂O (0.25 mL) and t-BuOH(0.25 mL). To this solution was added 0.1 M Sodium Ascorbate (70 μL,0.007 mmol, 1.0 equiv.), and 0.1 M copper(II) sulfate (35 μL, 0.0035mmol, 0.5 equiv.). The vial was sealed and subjected to microwaveirradiation for 1 hour at 110° C., after which the reaction wasconcentrated to yield a crude, dark red oil. The oil was taken up with67% TFA in CH₂Cl₂ (3 mL) into a 5 mL vial, and the vial was subjected tomicrowave irradiation for 2 minutes at 70° C. The reaction wasconcentrated down, taken up with 50% MeCN in H₂O, and purified by HPLC(Sunfire™ Prep C18 column (10×150 mm) using a 50% MeCN to 80% MeCN inH₂O gradient over 66 min at 5 mL/min). The product was isolated andlyophilized to yield s-22 as a white powder (3 mg, 15%). IR (thin film)3296 (br), 2934 (m), 2864 (w), 1643 (s), 1556 (m), 1463 (w), 1202 (m),1134 (m) cm-1. 1HNMR (500 MHz, MeOD) d 8.70 (s, 1H), 8.48 (d, J=1.5 Hz,2H), 8.38 (t, J=1.5 Hz, 1H), 7.86 (s, 2H), 4.78 (s, 2H), 4.42 (d, J=3.2Hz, 4H), 4.39 (t, J=7.2 Hz, 4H), 4.30 (dd, J=5, 9 Hz, 2H), 4.27 (dd,J=5, 8.5 Hz, 2H), 3.77-3.75 (m, 4H), 3.72-3.60 (m, 32H), 3.47 (t, J=7.2Hz, 4H), 3.15 (t, J=7.2 Hz, 22H), 2.43-2.39 (m, 4H), 2.24-2.14 (m, 26H),1.94-1.84 (m, 10H), 1.70-1.64 (m, 12H), 1.63-1.55 (m, 20H), 1.53-1.37(m, 30H), 1.35-1.29, (m, 20H). HRMS (ES+) calc'd for C₁₂₉H₂₁₈N₂₈O₃₆(M+H) m/z 2737.6191. Found 2737.6153.

(S)-bis((9H-fluoren-9-yl)methyl)(6-oxo-6-(prop-2-yn-1-ylamino)hexane-1,5-diyl)dicarbamate (s-23)

To a solution of Fmoc-Lys(Fmoc)-OH (1 g, 1.69 mmol, 1.0 equiv.) inCH₂Cl₂ (30 mL) was added EDC.HCl (391 mg, 2.03 mmol, 1.2 equiv.),HOBt.H₂O (311 mg, 2.03 mmol, 1.2 equiv.), followed by propargylamine (93mg, 0.11 mL, 1.69 mmol, 1.0 equiv.) and DIPEA (264 mg, 0.36 mL, 2.03mmol, 1.2 equiv.). The reaction was allowed to stir at room temperaturefor 90 minutes, upon which the reaction turned into a gel. The gel wasbroken up with a spatula and taken up with CH₂Cl₂ (50 mL). The mixturewas sonicated and filtered. The filtrate was collected, washed with 10%citric acid (50 mL), saturated sodium bicarbonate (50 mL), and brine (50mL). The organic layer was collected, dried with MgSO₄, and concentratedunder reduced pressure to give s-23 as a white powder (330 mg, 31%yield). IR (thin film) 3297 (s), 3068 (br), 2937 (br), 1687 (s), 1650(s), 1539 (m), 1450 (w), 1264 (w) cm-1. 1HNMR (500 MHz, CDCl3) d 7.75(t, J=7.8 Hz, 4H), 7.56 (d, J=7.4 Hz, 4H), 7.39 (q, J=7.5 Hz, 4H), 7.30(q, J=7.5 Hz, 4H), 6.28 (bs, 1H), 5.44 (bs, 1H), 4.85 (bs, 1H),4.43-4.35 (m, 4H), 4.21-4.30 (m, 3H), 4.02 (s, 2H), 3.25-3.15 (m, 2H),2.18 (t, J=2.4 Hz, 1H), 1.95-1.85 (m, 1H), 1.75-1.65 (m, 1H), 1.60-1.50(m, 2H), 1.42-1.32 (m, 2H). 13CNMR (125 MHz, CDCl3) d 171.4, 156.9,144.1, 144.0, 143.9, 143.8, 141.5, 141.4, 127.9, 127.8, 127.2, 127.2,125.1, 125.1, 120.2, 120.1, 79.3, 72.0, 67.2, 66.8, 47.4, 40.3, 31.7,29.6, 29.4, 22.3. HRMS (ES+) calc'd for C₃₉H₃₇N₃O₅ (M+H) m/z 628.2806.Found 628.2802.

(S)-2,6-diamino-N-(prop-2-yn-1-yl)hexanamide (s-24)

s-23 (300 mg) was dissolved in 10 mL of Et₂NH and 10 mL of CH₂Cl₂. Thereaction was allowed to stir for 24 hours, after which it wasconcentrated and purified with column chromatography (25% MeOH inCH₂Cl₂, then 25% MeOH in CH₂Cl₂+2% NH₄OH) to yield s-24 as a white solid(48 mg, 55%). IR (thin film) 3278 (br), 2931 (m), 2861 (w), 1649 (s),1554 (s), 1345 (w), 1262 (w), 920 (w) cm-1. 1HNMR (400 MHz, MeOD) d 3.96(dd, J=2.4, 7.2 Hz, 2H), 3.26 (t, J=7 Hz, 1H), 2.71 (t, J=7.6 Hz, 2H),2.59 (m, 1H), 1.69-1.61 (m, 1H), 1.58-1.47 (m, 3H), 1.46-1.34 (m, 2H).13CNMR (100 MHz, MeOD) d 177.3, 80.1, 72.3, 55.9, 41.7, 36.0, 31.8,29.3, 23.8. HRMS (ES+) calc'd for C₉H₁₇N₃O (M+H) m/z 184.1444. Found184.1441.

(S)-bis((9H-fluoren-9-yl)methyl)(11,19-dioxo-13-(prop-2-yn-1-ylcarbamoyl)-3,6,9,21,24,27-hexaoxa-12,18-diazanonacosane-1,29-diyl)dicarbamate(s-25)

Fmoc-miniPEG3-COOH (164 mg, 0.38 mmol, 2.2 equiv.) was dissolved inCH2Cl2 (5 mL). To this solution was added EDC.HCl (84 mg, 0.44 mmol, 2.5equiv.) and HOBt.H2O (67 mg, 0.44 mmol, 2.5 equiv.). The resultingsolution was transferred to a flask containing s-24 (32 mg, 0.174 mmol,1.0 equiv.). DIPEA (68 mg, 92 □L, 0.52 mmol, 3.0 equiv.) was added,followed by 1 mL of DMF. The reaction was allowed to stir at roomtemperature for 6 hours, after which it was concentrated andchromatographed (5% MeOH in CHCl₃) to yield a clear oil (122 mg, 70%).IR (thin film) 3307 (br), 3063 (w), 2918 (s), 1712 (m), 1659 (m), 1535(s), 1450 (w), 1253 (m), 1105 (m) cm-1. 1HNMR (500 MHz, CDCl3) d 7.76(d, J=7.5 Hz, 4H), 7.60 (d, J=7.5 Hz, 4H), 7.39 (t, J=7.5 Hz, 4H), 7.30(t, J=7.5 Hz, 4H), 6.97 (bs, 1H), 6.84 (bs, 1H), 5.74 (bs, 1H), 5.63(bs, 1H), 4.44-4.38 (m, 5H), 4.21 (t, J=7 Hz, 2H), 4.05-3.96 (m, 6H),3.65-3.61 (m, 16H), 3.57-3.55 (m, 4H), 3.42-3.35 (m, 4H), 3.28-3.21 (m,3H), 2.15 (t, J=2 Hz, 1H), 1.93-1.87 (m, 1H), 1.72-1.66 (m, 1H),1.56-1.50 (m, 2H), 1.39-1.34 (m, 2H). 13CNMR (125 MHz, CDCl3) d 171.2,170.6, 170.1, 156.8, 144.2, 144.1, 141.5, 127.8, 127.8, 127.2, 125.3,125.2, 120.1, 71.7, 71.1, 71.0, 70.6, 70.5, 70.5, 70.4, 70.4, 70.2,70.2, 66.7, 66.7, 52.5, 47.4, 47.4, 41.1, 38.6, 31.5, 29.2, 23.1. HRMS(ES+) calc'd for C₅₅H₆₇N₅O₁₃ (M+H) m/z 1007.4840. Found 1007.4839.

(S)—N,N′-(6-oxo-6-(prop-2-yn-1-ylamino)hexane-1,5-diyl)bis(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)acetamide)(s-26)

s-25 (120 mg) was dissolved in 5 mL of CH₂Cl₂ and 5 mL of Et₂NH. Thereaction was allowed to stir at room temperature for 24 hours, afterwhich it was concentrated and chromatographed (1×15 cm silica gel, 25%MeOH in CH₂Cl₂, then 25% MeOH in CH₂Cl₂+2% NH₄OH) to yield a colorlessoil (42 mg, 63%). IR (thin film) 3289 (br), 2865 (m), 1655 (s), 1532(m), 1457 (w), 1103 (m) cm-1. 1HNMR (400 MHz, MeOD) d 4.40 (dd, J=5.4,8.8 Hz, 1H), 4.04 (s, 2H), 3.98-3.95 (m, 4H), 3.73-3.61 (m, 16H), 3.53(dt, J=2, 5.2 Hz, 4H), 3.24 (t, J=7 Hz, 2H), 2.62 (t, J=1.5 Hz, 1H),1.89-1.80 (m, 1H), 1.76-1.68 (m, 1H), 1.59-1.52 (m, 2H), 1.43-1.33 (m,2H). 13CNMR (100 MHz, MeOD), d 173.5, 172.6, 172.6, 80.5, 73.3, 73.2,72.4, 72.0, 71.9, 71.6, 71.5, 71.4, 71.4, 71.2, 71.2, 71.2, 53.9, 42.1,42.1, 39.7, 33.1, 30.1, 29.5, 24.1. HRMS (ES+) calc'd for C₂₅H₄₇N₅O₉(M+H) m/z 562.3447. Found 562.3448.

s-27

s-21 (162 mg, 0.091 mmol, 1 equiv.) and s-26 (34 mg, 0.091 mmol, 1equiv.) was dissolved in H₂O (1.5 mL) and t-BuOH (1.5 mL) in a 5 ml,microwave vial. To this solution was added 0.1 M sodium ascorbate (0.6mL, 1 equiv.) and 0.1 M copper(II) sulfate (0.3 mL, 0.5 equiv.). Thevial was sealed and subjected to microwave irradiation for 1 hour at110° C., after which the reaction was concentrated under reducedpressure to yield a crude, brown oil. The crude oil was taken up with67% TFA in CH₂Cl₂ (3 mL) in a 5 mL microwave vial. The vial was sealedand subjected to microwave irradiation for 2 minutes at 70° C., afterwhich the reaction was concentrated under reduced pressure and purifiedusing HPLC (Sunfire™ Prep C18 column (10×150 mm) using a 50% MeCN to 80%MeCN in H₂O gradient over 66 min at 5 mL/min). The product was isolatedand lyophilized to yield a white powder (25 mg, 14%, 2 steps). IR (thinfilm) 3300 (br), 3093 (w), 2935 (m), 2866 (w), 1640 (s), 1552 (m), 1459(w), 1201 (w), 1135 (w) cm-1. 1HNMR (500 MHz, MeOD) d 8.56 (s, 1H), 8.45(d, J=1.6 Hz, 2H), 8.37 (t, J=1.5 Hz, 2H), 7.86 (s, 2H), 5.49 (s, 2H),4.58 (s, 2H), 4.45-4.40 (m, 6H), 4.39 (t, J=7 Hz, 4H), 4.30 (dd, J=5, 9Hz, 2H), 4.26 (dd, J=5, 9 Hz, 2H), 4.07 (s, 2H), 4.00 (s, 2H), 3.73-3.66(m, 24H), 3.43 (t, J=7 Hz, 4H), 3.23 (dt, J=3.1, 7.5 Hz, 2H), 3.17-3.13(m, 26H), 2.43-2.39 (m, 4H), 2.24-2.13 (m, 28H), 1.96-1.82 (m, 10H),1.78-1.71 (m, 2H), 1.71-1.63 (m, 12H), 1.63-1.54 (m, 24H), 1.53-1.46 (m,24H), 1.44-1.37 (m, 12H), 1.35-1.29 (m, 22H), 1.04 (d, J=5.6 Hz, 2H).13CNMR (125 MHz, MeOD) d 176.3, 176.1, 175.9, 175.8, 174.1, 172.6,172.5, 171.1, 167.8, 161.5, 161.2, 160.1, 147.4, 138.6, 138.3, 127.9,124.3, 122.7, 122.6, 118.4, 116.1, 71.8, 71.7, 71.4, 71.2, 71.2, 71.1,67.9, 67.9, 54.8, 54.2, 53.7, 53.5, 51.1, 41.1, 40.7, 40.6, 40.2, 39.6,37.0, 36.8, 35.6, 32.9, 32.8, 31.1, 30.7, 30.1, 30.1, 28.8, 27.6, 27.5,26.7, 26.5, 25.7, 24.1, 23.4, 17.6, 12.9. HRMS (ES+) calc'd forC₁₃₅H₂₂₈N₃₂O₃₇ (M+3H) m/z 964.2387. Found (M+3H) m/z 964.2380.

Bis(cp33)-Bis(Ac6-Urea)

s-27 (1.0 mg, 0.31 pmol, 1.0 equiv.) was dissolved in 0.15 mL of DMF. Tothis solution was added a solution of cp33-Ac—NHS (1.48 mg, 0.62 □mol,2.0 equiv.) in 0.15 mL of DMF. DIPEA was subsequently added, and thereaction was allowed to stir at room temperature for 12 hours. Thereaction was quenched with 1 mL of water and injected directly onto theHPLC and purified (Sunfire™ Prep C18 column (10×150 mm) using a 20% MeCNto 60% MeCN in H₂O gradient over 30 min at 5 mL/min) to give a whitepowder (0.4 mg, 19%).

Biological Evaluation

The following buffers, solutions, proteins, antibodies, reagents,equipment, materials, software, etc. as indicated, were used in carryingout the biological evaluation.

Buffers and Solutions:

Ultra-low IgG FBS

Invitrogen #16250-086

RPMI-1640 medium

Invitrogen #11875-093

stored at 4° C.

RPMI-1640 phenol free media

Invitrogen #11835-030

RPMI Growth media

RPMI Medium 1640 FROM xxx

DMEM-hi glucose

XXXX

Color-Free ADCP Media

RPMI Medium 1640, liquidATCC #11835-030 without phenol red supplemented with 10% HI-FBSultra-low IgG and 1% penicillin-streptomycin

DPBS Solution

Invitrogen #14190-144

EDTA Detachment Solution

210 mL DPBS

392 mg EDTA disodium salt (5.0 mM)

84 mg EGTA (1.0 mM)

to pH 7.40.22 μM sterile filter

TBS-A Tris-buffered saline with 1.5% BSA

Proteins, Antibodies and Reagents

Avi-tagged recombinant human PSMA protein kindly provided by Dr. JanKonvalinka and Dr. Ceril Barinka

with reference

Anti-Dinitrophenyl-KLH Rabbit IgG Fraction

Invitrogen #A6430

The purchased solution was stored at 4° C.

Streptavidin-Alexa467 (Invitrogen)

Penicillin-Streptomycin, Liquid (10,000 units penicillin; 10,000 μgStreptomycin/mL)

Invitrogen #15140-163

Recombinant Human IFN-γ

Cell signaling technologies

#8901SC

Vybrant DiD Cell Labeling Solution

Invitrogen #V-22887

1 mM in ethanolFL-4 fluorophore

Vybrant DiO Cell Labeling Solution

Invitrogen #V-22886 1 mM in DMF

FL-1 fluorophore

Trypan Blue Stain 0.4%

Invitrogen #15250

AT10 antibody ab23336 lot #902047

Anti-PSMA antibody phycoerthyrin conjugated

From Abcam #AB-77228

Phycoerythrin calibration beads

Bangs Laboratories Quantum™ R-PE MESF (#827) Equipment, Materials andSoftware

96 Well Flat Bottom Immuno Plate

MaxiSorp, Non Sterile, PS Nunc #442404

C6 Flow Cytometer Accuri

with CFlow Plus software

Amnis Imagestream X flow cytometer with IDEAS analysis software

FlowJo software

Petri Dishes

BD Falcon #351029

100×15 mm

Tissue culture dish

BD Falcon #353003

100×20 mm

Synergy 2 Multimode Microplate Reader

BioTek with Gen 5 software

T-Flasks

BD Falcon #353136

75 cm² tissue culture treated

Streptavidin labeled 6 uM beads

Polysciences 6 uM YG fluoresebrite beads

0.7 ug/mL biotin loading lot #601514

1.4 ug/mL biotin loading lot #573565

6 uM non-fluorescent beads

2.0 ug/mL biotin loading lot #621277

Lucigenin Tokyo Chemicals

Prism graphpad software

Cell Culture General Procedures:

Cell Counting: A cell suspension (10 μL) was diluted in Trypan blue(0.4%, 90 μL). 10 μL of this mixture was loaded onto a hemocytometer.Live cells were counted visually under 10× magnification.

EDTA Detachment: Adherent cells were aspirated and washed with DPBS (5mL). To the flask was added the EDTA detachment solution (5 mL). Theflask was incubated (15 min). Cells were fully detached by gentlyrinsing the solution over the bottom of the flask. The cell suspensionwas pelleted, aspirated, suspended in media, and split as desired intonew flasks.

Incubations were done at 37° C. in a humidified atmosphere supplementedwith 5% CO2.

Pelleting was done by centrifuge for 5 minutes at 1100 rpm.

Cell Lines:

All cell lines were grown in an incubator (37° C.) supplemented with 5%CO2. Media was changed approximately every 4 days. Cells were splitapproximately 4:1. Cells were not grown beyond approximately 30passages.

U937 Cells were purchased from ATCC (#CRL-1593.2), grown in Petri dishesas a suspension with RPMI-1640 medium supplemented with 10% HI-FBS and1% penicillin-streptomycin.

IIA1.6 cells kindly provided by Dr. Van de Winkel J. G. J. and Dr.Leusen J. H. W, grown in tissue culture treated dishes as weaklyadherent cells with RPMI-1640 medium supplemented with 10% HI-FBS and 1%penicillin-streptomycin.

RM1.PGLS kindly provided by Dr. Michael Sadelain of Memorial SloanKetering Cancer Center grown in tissue culture treated dishes asadherent cells with DMEM-high glucose supplemented with 10% HI-FBS and1% Penicillin-streptomycin

Bead Observed % Labeling Measured Calculated Phagocytosis Target(pmol/bead) PSMA/um² PSMA/um² (50 nM SyAM-P2) Beads 8.2 5577 46.2 5.73876 24.0 2.9 1972 10.1 RM1.PGLS 918 0

Bead Binding Assay for PSMA Binding Constant

The binding of each SyAm derivative to PSMA was evaluated on PSMA-coatedbeads. 6 μm streptavidin labeled beads (Polysciences) were incubatedwith 400 μg/ml recombinant avi-tagged-PSMA protein for 30 minutes. Beadswere washed twice with PBS, blocked with 1 mg/ml biotin for 30 minutesand then washed twice with TBS-A. 10⁵ beads were incubated with adilution series of SyAm ranging from 1 μM-100 pM. Streptavidin-Alexa467(Invitrogen) was added to a final concentration of 3.3 μM. Samples wereincubated on ice for 30 minutes, washed twice with DPBS, and evaluatedby an Accuri C6 flow cytometer. For binding to RM1.PGLS cells identicalconditions were followed substituting RM1.PGLS cells for beads.

EC₅₀ of PSMA Binding EC₅₀ FcyRI Binding Molecule (nM) (nM) SyAM-P1 40.7248.7 SyAM-P2 26 SyAM-P3 23.2 57.7

IIA1.6 FcγRI Expressing Cell Binding Assay

Peptide dilutions have been prepared in DMSO usually starting form 1 mMstock solution with further 10 fold dilution. Cells IIa1.6 stablytransfected with FcγRIA/γ-chain and non-transfected IIa1.6 cells used asisogenic negative control, were grown to approximately 1.5-2×10⁶cells/mL density, spun down and reconstituted in RPMI 1640 mediumwithout phenol red, supplemented with 10% FBS and Pen/Strep mixture tocell density 1×10⁶ cells/mL. A 100 uL aliquot of cells was furthertransferred into an eppendorf tube and left to cool on ice for 5-10minutes. Following this 1 uL of peptide dissolved in DMSO was added tothe cells, bringing final concentration of DMSO to 1%. After 1 hourincubation on ice with a peptide 5 uL of Streptavidin-Alexafluor 488 (2mg/mL) was added to the cells and left incubating on ice for 30-60minutes. After the incubation cells were washed two times with 1 mL ofcold RPMI1640 medium without phenol red, supplemented with 10% FBS andPen/Strep and finally washed once in 1 mL TBS Buffer (150 mM NaCl and 25mM Tris, pH 7.4). Cells were resuspended in residual TBS buffer (usually˜100 uL) and analyzed via flow cytometry with detection in the FL1channel.

PSMA Measurement

RM1.PGLS cells detached with enzyme free detachment solution with gentlepipetting up and down. Cells resuspended to a concentration of 1×10⁶ inPBS with 5% BSA. This cell solution was transferred in 100 uL aliquotsto eppendorf tubes and to the appropriate tubes 2 uL of phycoerythrinlabeled anti-CD32a antibody was added. Eppendorfs were incubated on icefor 30 minutes. Samples were centrifuged at 1.1 RPM for 5 minutes at 4cand washed 2× with PBS 5% BSA solution. Cells were run on Accuri C6 flowcytometer until 20 k live cell counts were achieved. QuantitationR-Phycoerythrin beads from Bangs Laboratories were run immediately aftersamples per manufacturers instructions. Geometric mean fluorescence ofFL-2 channel was entered into calibration worksheet provided by themanufacturer and PSMA levels calculated.

Effector Cell Priming

Three days before the experiments, a plate of U937 cells (approximately60% confluent) was passed into a new Petri dish (10 mL colored RPMIgrowth media total volume). IFN-γ (20 μL, 100 μg/mL in DPBS) was added,and the cells were maintained in an incubator (37° C., 24 h). The cellswere pelleted after 24 hours and resuspended in 10 mL of IFN-γ (20 uL,100 ug/mL in DPBS) and maintained in an incubator (37° C., 24 h)

For ROS production cells were resuspended in RPMI growth media (10 mL),and split equally into two Petri dishes. To each dish were addedadditional colored RPMI growth media (5 mL) and IFN-γ (20 μL, 100 μg/mLin DPBS), and maintained in an incubator (37° C., 24 h)

For phagocytosis: cells were transferred into a Falcon tube, and DiD (19uL, final concentration=1.9 μM) was added. The cells were maintained inan incubator (37° C., 30 min), pelleted, aspirated, resuspended in RPMIgrowth media (10 mL), and split equally into two Petri dishes. To eachdish were added additional colored RPMI growth media (5 mL) and IFN-γ(20 μL, 100 μg/mL in DPBS), and maintained in an incubator (37° C., 24h).

ROS Production Assay

Primed U937 were suspended in color-free ADCP media at a concentrationof 3×10⁵ U937 cells and mixed with 10⁵ PSMA coated beads and variousconcentrations of molecules in a 96 well plate in a volume of 90 uL. Toeach well was added 10 μl of 2.5 mM lucigenin (Tokyo Chemicals) solutionto bring the total volume to 1000. The plate was centrifuged at 200 rcffor 2 minutes (PLATE SPINNER). The chemiluminescence was then measuredat two minute intervals by plate reader (Biotek Synergy 2) for 60 to 90minutes.

Assay for Molecule Induced Phagocytosis of Beads

6 uM streptavidin fluoresbrite YG microspheres from Polysciences wereincubated with 4× concentration of avi-tagged PSMA for 30 minutes atroom temperature in DPBS (concentration of avi-tagged PSMA is dependenton biotin loading capacity of microspheres). Beads were washed with DPBS2× and resuspended in color-free ADCP media at 1.6 million beads/mL.

Phagocytosis: 25 uL of bead solution was added to 25 uL of ADCP mediaeither with or without molecules at various concentrations in aneppendorf tube. To this 50 uL of 4 million/mL primed U937 cells wereadded, gently mixed and then centrifuged at 1.1 RPM for 2 minutes. Thecaps were opened and placed in an incubator (37° C., 1 hr) at whichpoint the eppendorfs were closed and placed on ice. Samples werevortexed and run on an Accuri C6 flow cytometer for a total of 50 kcounts.

Data analysis: For each experiment 50,000 events were counted. Forwardand side scatter plots were optimally gated to remove debris particlesand cellular aggregates. On a plot of FL-2 vs FL-4 the followingpopulations were counted:

Population

Population Approximate FL-2 signal Approximate FL-4 signal EffectorCells 10³-10⁴   10⁵-10^(6.5) Target Beads 10⁶-10⁷ 10^(2.5)-10⁴   DoublePositive Cells 10⁶-10⁷   10⁵-10^(6.5)

${\% \mspace{14mu} {phagocytosis}} = {\frac{\left( {{double}\mspace{14mu} {positive}\mspace{14mu} {cells}} \right)}{\left( {{remaining}\mspace{14mu} {target}\mspace{14mu} {cells}} \right) + \left( {{double}\mspace{14mu} {positive}\mspace{14mu} {cells}} \right)} \times 100}$

Phagocytosis Assay of Cells

Target cell passing: Three days before the experiments, a plate oftarget cells, RM1.PGLS cells were passed into a new flask.

Effector cell priming: Three days before the experiments, a plate ofU937 cells (approximately 60% confluent) was passed into a new Petridish (10 mL colored ADCP media total volume). IFN-γ (20 μL, 100 μg/mL inDPBS) was added, and the cells were maintained in an incubator (37° C.,24 h). The cells were transferred into a Falcon tube, and DiD (19 uL,final concentration=1.9 μM) was added. The cells were maintained in anincubator (37° C., 30 min), pelleted, aspirated, resuspended in coloredADCP media (10 mL), and split equally into two Petri dishes. To eachdish were added additional colored ADCP media (5 mL) and IFN-γ (20 μL,100 μg/mL in DPBS), and the cells were maintained in an incubator (37°C., 24 h).

Target cell preparation: To a plate of target cells (60-80% confluentRM1.PGLS cells 10 mL total media volume) was added DiO (20 μL, finalconcentration=2 μM). The cells were maintained in an incubator (37° C.,30 min), aspirated, and washed with colored ADCP media (3×10 mL).Colored ADCP media (10 mL) was added. The cells were maintained in anincubator (37° C., 2 h), detached and resuspended in color-free ADCPmedia at a concentration of 0.5 million cells per mL.

Effector cell preparation: Both dishes of primed U937 cells weretransferred into a Falcon tube, pelleted, aspirated, resuspended incolor-free ADCP media (10 mL), counted, and diluted with color-free ADCPmedia to give a final concentration of 2 million cells per mL.

Phagocytosis: All conditions were run in triplicate. For eachexperiment, into a sterile 2 mL Eppendorf tube were added color-freeADCP media (25 μL) containing either various concentrations of SyAM-Pxor ARM-P8/Anti-DNP antibody, target cells (25 μL=12,500 cells), andeffector cells (50 μL=100,000 cells), to give an effector-to-targetratio of 8:1. Tubes were gently agitated by hand. The cells werepelleted (2 min, 1100 rpm). The tubes were opened, maintained in anincubator (37° C., 1 h), resuspended by briefly agitating with a vortex,and analyzed by flow cytometry.

Population Approximate FL-1 signal Approximate FL-4 signal EffectorCells 10³-10⁴   10⁵-10^(6.5) Target Cells 10⁶-10⁷ 10^(2.5)-10⁴   DoublePositive Cells 10⁶-10⁷   10⁵-10^(6.5)

${\% \mspace{14mu} {phagocytosis}} = {\frac{\left( {{double}\mspace{14mu} {positive}\mspace{14mu} {cells}} \right)}{\left( {{remaining}\mspace{14mu} {target}\mspace{14mu} {cells}} \right) + \left( {{double}\mspace{14mu} {positive}\mspace{14mu} {cells}} \right)} \times 100}$

Amnis Imagestream Imaging

The phagocytosis assay was performed as described above. After one hour,cells were fixed in 3% formaldehyde for 30 minutes on ice. Cells werewashed once in DPBS then stained with anti-CD14-APC and anti-CD11b-APCantibodies (Biolegend) for 30 minutes on ice. Cells were washed once inDPBS then passed through a 70 μm cell strainer. 30,000 events per samplewere collected on the Amnis Imagestream X flow cytometer. Doublepositive events were then manually scored for phagocytic cup formationor complete engulfment of the target. Data was analyzed on Amnis IDEASsoftware.

REFERENCES

-   1. Hansel, T. T.; Kropshofer, H.; Singer, T.; Mitchell, J. A.;    George, A. J. T., The safety and side effects of monoclonal    antibodies. Nat Rev Drug Discov 2010, 9 (4), 325-338.-   2. Weiner, L. M., Building better magic bullets-improving    unconjugated monoclonal antibody therapy for cancer. Nat Rev Cancer    2007, 7 (9), 701-706.-   3. Siberil, S.; Dutertre, C. A.; Fridman, W. H.; Teillaud, J. L., Fc    gamma R: The key to optimize therapeutic antibodies? Crit. Rev.    Oncol./Hematol. 2007, 62 (1), 26-33.-   4. McEnaney, P. J.; Parker, C. G.; Zhang, A. X.; Spiegel, D. A.,    Antibody-Recruiting Molecules: An Emerging Paradigm for Engaging    Immune Function in Treating Human Disease. ACS Chemical Biology    2012, 7 (7), 1139-1151.-   5. Cuesta, n. M.; Sainz-Pastor, N.; Bonet, J.; Oliva, B.;    _(i)lvarez-Vallina, L., Multivalent antibodies: when design    surpasses evolution. Trends in biotechnology 2010, 28 (7), 355-362.-   6. James, N. D.; Atherton, P. J.; Jones, J.; Howie, A. J.;    Tchekmedyian, S.; Cumow, R. T., A phase II study of the bispecific    antibody MDX-H210 (anti-HER2×CD64) with GM-CSF in HER2+ advanced    prostate cancer. Br J Cancer 2001, 85 (2), 152-156.-   7. Li, Y.; O'Dell, S.; Walker, L. M.; Wu, X.; Guenaga, J.; Feng, Y.;    Schmidt, S. D.; McKee, K.; Louder, M. K.; Ledgerwood, J. E.;    Graham, B. S.; Haynes, B. F.; Burton, D. R.; Wyatt, R. T.;    Mascola, J. R., Mechanism of Neutralization by the Broadly    Neutralizing HIV-1 Monoclonal Antibody VRC01. Journal of Virology    2011, 85 (17), 8954-8967.-   8. (a) Jakobsche, C. E.; McEnaney, P. J.; Zhang, A. X.; Spiegel, D.    A., Reprogramming Urokinase into an Antibody-Recruiting Anticancer    Agent. ACS Chemical Biology 2011, 7 (2), 316-321; (b) Murelli, R.    P.; Zhang, A. X.; Michel, J.; Jorgensen, W. L.; Spiegel, D. A.,    Chemical Control over Immune Recognition: A Class of    Antibody-Recruiting Small Molecules That Target Prostate Cancer.    Journal of the American Chemical Society 2009, 131 (47),    17090-17092; (c) Parker, C. G.; Domaoal, R. A.; Anderson, K. S.;    Spiegel, D. A., An Antibody-Recruiting Small Molecule That Targets    HIV gp120. Journal of the American Chemical Society 2009, 131 (45),    16392-16394.-   9. Spiegel, D. A., Grand Challenge Commentary: Synthetic immunology    to engineer human immunity. Nat Chem Biol 2010, 6 (12), 871-872.-   10. Society, A. C., Cancer Facts & Figures 2011. Society, A. C., Ed.    Atlanta, 2011.-   11. Nimmerjahn, F.; Ravetch, J. V., Fc[gamma] receptors as    regulators of immune responses. Nat Rev Immunol 2008, 8 (1), 34-47.-   12. Bonetto, S.; Spadola, L.; Buchanan, A. G.; Jermutus, L.; Lund,    J., Identification of cyclic peptides able to mimic the functional    epitope of IgG1-Fc for human Fc gamma RI. Faseb J. 2009, 23 (2),    575-585.-   13. Berntzen, G.; Andersen, J. T.; Ustgard, K.; Michaelsen, T. E.;    Mousavi, S. A.; Qian, J. D.; Kristiansen, P. E.; Lauvrak, V.;    Sandlie, I., Identification of a High Affinity Fe gamma RIIA-binding    Peptide That Distinguishes Fc gamma RBA from Fc gamma RIIB and    Exploits Fc gamma RBA-mediated Phagocytosis and Degradation. J.    Biol. Chem. 2009, 284 (2), 1126-1135.-   14. Cendron, A. C.; Wines, B. D.; Brownlee, R. T. C.; Ramsland, P.    A.; Pietersz, G. A.; Hogarth, P. M., An FcγRIIa-binding peptide that    mimics the interaction between FcγRIIa and IgG. Molecular Immunology    2008, 45 (2), 307-319.-   15. Wang, S.-Y.; Veeramani, S.; Racila, E.; Cagley, J.;    Fritzinger, D. C.; Vogel, C.-W.; St John, W.; Weiner, G. J.,    Depletion of the C3 component of complement enhances the ability of    rituximab-coated target cells to activate human NK cells and    improves the efficacy of monoclonal antibody therapy in an in vivo    model. Blood 2009, 114 (26), 5322-5330.

1. A compound according to the chemical structure:

where [IBT] is an FcγRI receptor binding moiety; [CBT] is a cell bindingmoiety which binds to prostate specific membrane antigen (PSMA); L₁ andL₂ are linker groups, which groups optionally include one or morebifunctional connector groups [CON}; [MULTICON} is a bifunctional ormultifunctional connector group which, when present, connects at leastone [IBT) group to at least one [CBT] group through a linker; MCON is aninteger from 0 to 10; NL1 and NL2 are each an integer from 0 to 10, withthe proviso that n≧NL1 and n′≧NL2.
 2. The compound according to claim 1wherein MCON is 1, 2 or
 3. 3. The compound according to claim 1 whereinMCON is
 1. 4. The compound according to claim 1 wherein n is 1, 2 or 3and n″ is 1 or
 2. 5. The compound according to claim 1 wherein NL1 is 1and NL2 is
 1. 6. The compound according to claim 1 wherein MCON is 0,NL1 is 1 and NL2 is
 1. 7. The compound according to claim 1 wherein MCONis 0, n is 1 and n′ is
 1. 8. The compound according to claim 1 whereinNL1 is
 1. 9. The compound according to claim 1 wherein NL2 is
 1. 10. Thecompound according to claim 1 wherein said [IBT] is a group as set forthin attached FIG.
 14. 11. The compound according to claim 1 wherein said[IBT] group is CP33.
 12. The compound according to claim 1 wherein said[IBT] group is CP33.
 13. The compound according to claim 1 wherein said[CBT] group is a group according to the chemical structure:

Where X₁ and X₂ are each independently CH₂, O, NH or S; X₃ is O, CH₂,NR¹, S(O), S(O)₂, —S(O)₂O, —OS(O)₂, or OS(O)₂O; R¹ is H, a C₁-C₃ alkylgroup, or a —C(O)(C₁-C₃) group; k is an integer from 0 to 20, or a saltor enantiomer thereof.
 13. (canceled)
 14. The compound according toclaim 60 wherein k is
 4. 15. The compound according to claim 1optionally containing a [CON] group according to the chemical structure:

Where X² is O, S, NR⁴, S(O), S(O)₂, —S(O)₂O, —OS(O)₂, or OS(O)₂O; X³ isO, S, NR⁴; and R⁴ is H, a C₁-C₃ alkyl or alkanol group, or a—C(O)(C₁-C₃) group.
 16. The compound according to claim 1 containing a[CON] group wherein said [CON] group is according to the chemicalstructure;

Where CL is

m is an integer from 0 to 12, often 0, 1, 2, 3, 4, 5, 6 and iL is 0 or1, often
 1. 17. The compound according to claim 1 wherein said linkergroup L₁ or L₂ comprises polyethyleneglycol (PEG) linkages,polypropylene glycol linkages, or polyethyleneglycol-co-polypropylenepolymers from 1 to 100 units in length; A polyproline linkers orcollagen linker according to the chemical structure

where n is 1 to 100; a linker according to the structure:

Where R_(a) is H, C₁-C₃ alkyl, alkanol or forms a cyclic ring with R³ inthe case of proline and R³ is a side chain derived from an amino acid;m″ is an integer from 0 to 25; and m is an integer from 1 to 100;wherein each of said groups may be further linked through amide groups,keto groups, amine groups or amino acids; or a linker according to thestructure:

where R_(a) is H or a C₁-C₃ alkyl, preferably CH₃, most often H; m is aninteger from 1 to 12, often 1, 2, 3, 4, 5, or 6; m″ is an integer 1, 2,3, 4, 5, or 6, often 6; t is 0, 1, 2, 3, 4, 5, or 6; and iL is 0 or 1,wherein said linker is optionally linked to a [CON] group and a [CBT]group at one end and a [MULTICON] group on the other end; or a linkeraccording to the structure:

Where q is an integer from 0-12; and q′ is 1 to 12; or a linkeraccording to the chemical structure:

Where q is an integer from 0-12; and q′ is 1 to 12; iL is 0 or 1; andR_(L) is an amino acid or an oligopeptide or a linker succinimideaccording to the chemical structure:

where each X^(S) is independently S, O or N—R^(S); R^(S) is H or C₁₋₃alkyl; S_(c) is CH₂; CH₂O; or CH₂CH₂O; i is 0 or 1; and m^(S) is 0, 1,2, 3, 4, 5, or 6, or a linker according to the chemical formula:

Where Z and Z′ are each independently a bond, —(CH₂)_(i)—O,—(CH₂)_(i)—S, —(CH₂)_(i)—N—R,

wherein said Z or Z′ group, is optionally bonded to another linkergroup, a connector group [CON], a [MULTICON] group, IBT or CBT; each Ris H, or a C₁-C₃ alkyl or alkanol group; each R² is independently H or aC₁-C₃ alkyl group; each Y is independently a bond, O, S or N—R; each iis independently 0 to 100; D is

or a bond, or D may be

or a polypropylene glycol or polypropylene-co-polyethylene glycol linkerhaving between 1 and 100 glycol units; with the proviso that Z, Z′ and Dare not each simultaneously bonds; each i is the same as above; j is 1to 100; m (within this context) is an integer from 1 to about 100; and n(within this context) is an integer from 1 to about 100; m′ is 1 to 100;m″ is an integer between 0 to 25, preferably 1 to 10, 1 to 8, 1, 2, 3,4, 5, or 6; n′ is 1 to 100, 1 to 75, 1 to 60, 1 to 55, 1 to 50, 1 to 45,1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1, 2, 3, 4or 5; X¹ is O, S or N—R; R is as described above; R_(a) is H, C₁-C₃alkyl or alkanol or forms a cyclic ring with R³; and R³ is a side chainderived from an amino acid.
 18. The compound according to claim 1wherein said [MULTICON] group is a multifunctional connector group ormolecule according to the chemical structure:

where Y₄ is C—H or N; and Each X″ is independently derived from anelectrophilic or nucleophilic group, preferably (CH₂)_(n″)O,(CH₂)_(n″)N^(RCON), (CH₂)_(n″)S, (CH₂)_(n″), (CH₂)_(n″)C═O or a [CON]group; the substitutent RCON is H or a C₁-C₃ alkyl, preferably H or CH₃and n″ is 0, 1, 2 or 3; r is an integer from 1 to 12; and said [CON]group, if present, is a moiety according to the chemical structure:

Where X² is O, S, NR⁴, S(O), S(O)₂, —S(O)₂O, —OS(O)₂, or OS(O)₂O; X³ isNR⁴, O or S; and R⁴ is H, a C₁-C₃ alkyl or alkanol group, or a—C(O)(C₁-C₃) group; or a pharmaceutically acceptable salt thereof. 19.The compound according to claim 1 wherein L₁ and/or L₂ is a

group where R_(a) is H or CH₃; m is an integer from 1 to 12; m″ is aninteger 1, 2, 3, 4, 5, or 6; t is 0, 1, 2, 3, 4, 5, or 6; and IL is 0or
 1. 20. The compound according to claim 1 wherein L₁ and/or L₂ is

where R_(a) is H; m″ is 1, 2, 3, 4, 5 or 6 and m is 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11 or
 12. 21. The compound according to claim 17 wherein L₁and/or L₂ is a polyethylene glycol linker between 1 and 12 glycol unitsin length or a polyethylene glycol linker extended to a secondpolyethylene glycol linker through a [CON] group, wherein saidpolyethylene glycol linker is from 1 to 12 glycol units in length andsaid second polyethylene glycol linker is from 1 to 12 glycol units inlength.
 22. The compound according to claim 21 wherein said [CON] groupis


23. The compound according to claim 1 which is the compound FIG. 16 or apharmaceutically acceptable salt, stereoisomer, solvate or polymorphthereof.
 24. The compound of FIG. 16 wherein said biotin group isabsent.
 25. The compound of claim 23 wherein said lysine group attachedto said biotin group is replaced with an amino acid having no functionalgroup in the amino acid sidechain.
 26. The compound according to claim 1which is compound 2 of FIG. 2 or a pharmaceutically acceptable salt,stereoisomer, solvate or polymorph thereof.
 27. The compound accordingto claim 26 wherein said biotin is absent.
 28. The compound according toclaim 26 wherein said lysine group attached to said biotin group isreplaced with an amino acid having no functional group in the amino acidsidechain.
 29. The compound according to claim 1 which is compound 3 ofFIG.
 2. 30. A pharmaceutical composition comprising an effective amountof a chimeric compound according to claim 1 in combination with apharmaceutically acceptable carrier, additive or excipient.
 31. Thecomposition according to claim 30 wherein said composition furthercomprises an effective amount of an additional anticancer agent.
 32. Thecomposition according to claim 31 wherein said additional anticanceragent is an antimetabolite, an inhibitor of topoisomerase I and II, analkylating agent, a microtubule inhibitor or mixtures thereof.
 33. Thecomposition according to claim 31 wherein said agent is aldesleukin;aemtuzumab; alitretinoin; allopurinol; altretamine; amifostine;anastrozole; arsenic trioxide; aparaginase; BCG Live; bexarotenecapsules; bexarotene gel; bleomycin; busulfan intravenous; busulfanoral; calusterone; capecitabine; carboplatin; carmustine; carmustinewith poifeprosan 20 iplant; celecoxib; chlorambucil; cisplatin;cladribine; cyclophosphamide; cytarabine; cytarabine liposomal;dacarbazine; dactinomycin; actinomycin D; dabepoetin alfa; daunorubicinliposomal; daunorubicin, daunomycin; dnileukin diftitox, dexrazoxane;docetaxel; doxorubicin; doxorubicin liposomal; domostanolone propionate;eliott's B soution; epirubicin; eoetin alfa estramustine; etoposidephosphate; etoposide (VP-16); exemestane; flgrastim; floxuridine(intraarterial); fludarabine; fluorouracil (5-FU); fulvestrant;gemtuzumab ozogamicin; goserelin acetate; hydroxyurea; IbritumomabTiuxetan; idarubicin; ifosfamide; imatinib mesylate; Interferon alfa-2a;Interferon alfa-2b; irinotecan; letrozole; leucovorin; levamisole;lomustine (CCNU); meclorethamine (nitrogen mustard); megestrol acetate;melphalan (L-PAM); mercaptopurine (6-MP); mesna; methotrexate;methoxsalen; mitomycin C; mitotane; mitoxantrone; nandrolonephenpropionate; nfetumomab; LOddC; orelvekin; oxaliplatin; paclitaxel;pamidronate; pegademase; Pegaspargase; Pegfilgrastim; pentostatin;pipobroman; plicamycin; mithramycin; porfimer sodium; procarbazine;quinacrine; Rasburicase; Rituximab; Sargramostim; streptozocin;talbuvidine (LDT); talc; tamoxifen; temozolomide; teniposide (VM-26);testolactone; thioguanine (6-TG); thiotepa; topotecan; toremifene;Tositumomab; Trastuzumab; tretinoin (ATRA); Uracil Mustard; valrubicin;valtorcitabine (monoval LDC); vinblastine; vinorelbine; zoledronate; andmixtures thereof.
 34. The composition according to claim 30 furthercomprising at least one antiandrogen compound.
 35. The compositionaccording to claim 30 further comprising at least one GNRh modulator.36. The composition according to claim 30 further comprising at leastone agent selected from the group consisting of flutamide, bicalutamide,nilutamide, cyproterone acetate, ketoconazole, aminoglutethimide,abarelix, leuprolide, goserelin, triptorelin, buserelin, abirateroneacetate, sorafenib and mixtures thereof.
 37. The composition accordingto claim 30 further comprising at least one agent selected from thegroup consisting of an enlarged prostate agent, eulexin, flutamide,goserelin, leuprolide, lupron, nilandron, nilutamide, zoladex andmixtures thereof.
 38. The composition according to claim 30 in oraldosage form.
 39. The composition according to claim 30 in parenteraldosage form.
 40. The composition according to claim 39 wherein saidparenteral dosage form is an intravenous dosage form.
 41. Thecomposition according to claim 30 in topical dosage form.
 42. A methodof treating prostate cancer in a patient in need thereof comprisingadministering to said patient an effective amount of a compoundaccording to claim
 1. 43. The method according to claim 42 wherein saidprostate cancer is metastatic prostate cancer.
 44. A method of treatingprostate cancer in a patient in need thereof comprising administering tosaid patient an effective amount of a composition according to claim 30.45. A method of inhibiting metastasis of prostate cancer in a patient inneed thereof comprising administering to said patient an effectiveamount of a compound according to claim 30 to said patient.
 46. A methodof treating cancer in a patient in need thereof comprising administeringto said patient a composition according to claim
 30. 47. The methodaccording to claim 46 wherein said cancer is stomach, colon, rectal,liver, pancreatic, lung, breast, cervix uteri, corpus uteri, ovary,testis, bladder, renal, brain/CNS, head and neck, throat, Hodgkin'sdisease, non-Hodgkin's lymphoma, multiple myeloma, leukemia, melanoma,non-melanoma skin cancer, acute lymphocytic leukemia, acute myelogenousleukemia, Ewing's sarcoma, small cell lung cancer, choriocarcinoma,rhabdomyosarcoma, Wilms' tumor, neuroblastoma, hairy cell leukemia,mouth/pharynx, oesophagus, larynx, kidney cancer or lymphoma.
 48. Amethod of treating prostate cancer in a patient wherein said patientalso has another form of cancer said method comprising administering tosaid patient an effective amount of a composition according to claim 30.49. The method according to claim 48 wherein said other form of canceris stomach, colon, rectal, liver, pancreatic, lung, breast, cervixuteri, corpus uteri, ovary, testis, bladder, renal, brain/CNS, head andneck, throat, Hodgkin's disease, non-Hodgkin's lymphoma, multiplemyeloma, leukemia, melanoma, non-melanoma skin cancer, acute lymphocyticleukemia, acute myelogenous leukemia, Ewing's sarcoma, small cell lungcancer, choriocarcinoma, rhabdomyosarcoma, Wilms' tumor, neuroblastoma,hairy cell leukemia, mouth/pharynx, oesophagus, larynx, kidney cancer orlymphoma.
 50. The method according to claim 48 wherein said other formof cancer is a carcinoma, a malignant melanoma, a myeloproliferativedisease, a sarcoma, a tumor of the central nervous system, a germ-linetumor, a mixed type of neoplasia or a tumor of mixed origin. 51.-59.(canceled)
 60. The compound according to claim 1 wherein said CBT groupis the group

Where k is 2, 3 or 4 or a salt thereof.